ELEMENTS 

OF 

WATER     BACTERIOLOGY 


WITH    SPECIAL     REFERENCE    TO 


SANITARY  WATER  ANALYSIS 


BY 

SAMUEL  GATE  PRESCOTT 

Associate  Professor  of  Industrial  Microbiology   in  the  Massachusetts 

Institute  of  Technology 

AND 

CHARLES-EDWARD  AMORY  WINSLOW 

Associate  Professor  of  Biology,    College  of  the   City  of  New   York,    and  Curator 
of  Public  Health,   American  Museum  of  Natural  History,  New   York 


THIRD  EDITION,  REWRITTEN 

FIRST    THOUSAND 


NEW  YORK 

JOHN  WILEY  &  SONS,  INC. 

LONDON:    CHAPMAN  &  HALL,  LIMITED 

1913 


QTK 1  0- 


CJDO  . 


Copyright,  1904,  1908,  1913 

BY 
:S.  C.  PRESCOTT  AND  C.-E.  A.  WINSLOW 


THE    SCIENTIFIC    PRESS 

ROBERT    DRUMMOND    AND    COMPANY 

BROOKLYN.    N.    V. 


DEDICATED 

TO 

William 


BY  TWO   OF  HIS   PUPILS, 
AS  A  TOKEN  OF  GRATEFUL  AFFECTION 


PREFACE   TO   FIRST   EDITION 


THE  general  awakening  of  the  community  to  the 
importance  of  the  arts  of  sanitation — accelerated  by  the 
rapid  growth  of  cities  and  the  new  problems  of  urban 
life — demands  new  and  accurate  methods  for  the  study 
of  the  microbic  world.  Bacteriology  has  long  since 
ceased  to  be  a  subject  of  interest  and  importance  to 
the  medical  profession  merely,  but  has  become  intimately 
connected  with  the  work  of  the  chemist,  the  biologist, 
and  the  engineer.  To  the  sanitary  engineer  and  the 
public  hygienist  a  knowledge  of  bacteriology  is  indis- 
pensable. 

In  the  swift  development  of  this  science  during  the 
last  ten  years  perhaps  no  branch  of  bacteriology  has 
made  more  notable  progress  than  that  which  relates  to 
the  sanitary  examination  of  water.  After  a  brief 
period  of  extravagant  anticipation,  and  an  equally 
unreasonable  era  of  neglect  and  suspicion,  the  methods 
of  the  practical  water  bacteriologist  have  gradually 
made  their  way,  until  it  is  recognized  that,  on  account 
of  their  delicacy,  their  directness,  and  their  certainty, 
these  methods  now  furnish  the  final  criterion  of  the 
sanitary  condition  of  a  potable  water. 


vi  PREFACE  TO  FIRST  EDITION 

A  knowledge  of  the  new  science  early  became  so 
indispensable  for  the  sanitary  expert  that  a  special 
course  in  the  Bacteriology  of  Water  and  Sewage  has  for 
some  years  been  given  to  students  of  biology  and  sani- 
tary engineering  in  the  Biological  Department  of  the 
Massachusetts  Institute  of  Technology.  For  workers 
in  this  course  the  present  volume  has  been  especially 
prepared,  and  it  is  fitting,  we  think,  that  such  a  manual 
should  proceed  from  an  institution  whose  faculty, 
graduates,  and  students  have  had  a  large  share  in  shap- 
ing the  science  and  art  of  which  it  treats.  We  shall 
be  gratified,  however,  if  its  field  of  usefulness  extends 
to  those  following  similar  courses  in  other  institutions, 
or  occupied  professionally  in  sanitary  work. 

The  treatment  of  the  subject  in  the  many  treatises 
on  General  Bacteriology  and  Medical  Bacteriology  is 
neither  special  enough  nor  full  enough  for  modern 
needs.  The  classic  work  of  Grace  and  Percy  Frank- 
land  is  now  ten  years  old;  and  even  Horrocks'  valuable 
"  Bacteriological  Examination  of  Water  "  requires  to 
be  supplemented  by  an  account  of  the  developments 
in  quantitative  analysis  which  have  taken  place  on  this 
side  of  the  Atlantic. 

It  is  for  us  a  matter  of  pride  that  Water  Bacteriology 
owes  much  of  its  value,  both  in  exactness  of  method 
and  in  common-sense  interpretation,  to  American 
sanitarians.  The  English  have  contributed  researches 
of  the  greatest  importance  on  the  significance  of  certain 
intestinal  bacteria;  but  with  this  exception  the  best 
work  on  the  bacteriology  of  water  has,  in  our  opinion, 
been  done  in  this  country.  Smith,  Sedgwick,  Fuller, 


PREFACE  TO  FIRST  EDITION  vii 

Whipple,  Jordan,  and  their  pupils  and  associates  (not 
to  mention  others)  have  been  pioneers  in  the  develop- 
ment of  this  new  field  in  sanitary  science.  To  gather 
the  results  of  their  work  together  in  such  form  as  to 
give  a  correct  idea  of  the  best  American  practice  is 
the  purpose  of  this  little  book;  and  this  we  have  tried 
to  do  with  such  completeness  as  shall  render  the  volume 
of  value  to  the  expert  and  at  the  same  time  with  such 
freedom  from  undue  technicality  as  to  make  it  reada- 
ble for  the  layman.  It  should  be  distinctly  understood 
that  students  using  it  are  supposed  to  have  had  before- 
hand a  thorough  course  in  general  bacteriology,  and 
to  be  equipped  for  advanced  work  in  special  lines. 
BOSTON,  March  10,  1904. 


PREFACE   TO   THIRD   EDITION 


A  SECOND  edition  of  this  work  was  called  for  in  1908 
and  it  was  rewritten  in  that  year,  with  the  inclusion 
of  much  new  material  in  the  chapters  dealing  with 
the  isolation  of  the  typhoid  bacillus  and  of  intestinal 
bacteria,  and  with  the  addition  of  a  new  chapter  on  the 
bacteriology  of  sewage  and  sewage  effluents.  In  the 
same  year  there  appeared  an  excellent  volume  on 
Water  Bacteriology  by  Dr.  W.  G.  Savage,  which  showed 
the  English  methods  of  investigation  and  interpre- 
tation to  be  closely  in  accord  with  those  used  in 
America. 

In  the  five  years  which  have  elapsed  since  our  second 
edition  was  published,  there  has  again  been  important 
progress  along  many  lines  in  sanitary  bacteriology; 
and  in  particular  the  publication  in  1912  of  a  new  edition 
of  the  Report  of  the  Committee  on  Standard  Methods 
of  the  Laboratory  Section  of  the  American  Public 
Health  Association  has  made  necessary  a  change  in 
many  details  of  current  practice. 

We  have,  therefore,  prepared  at  this  time  a  some- 
what far-reaching  revision  of  our  book.  Newer  ideas 

ix 


x  PREFACE  TO  THIRD  EDITION 

on  the  effect  of  temperature  upon  the  viability  of 
bacteria  in  water  are  included  in  Chapter  I.  The  recent 
recommendations  of  the  Committee  on  Standard 
Methods  are  discussed  in  Chapters  II  and  IV;  in 
particular,  Chapter  IV,  dealing  with  the  37°  count, 
has  been  expanded.  We  cannot  bring  ourselves  to 
agree  with  the  recommendation  of  the  committee  that 
the  37°  count  should  replace  the  20°  count;  but  we  are 
entirely  in  accord  with  the  resolution  adopted  by  the 
Laboratory  Section  of  the  American  Public  Health 
Association  at  its  Washington  meeting  that  both 
determinations  should  be  made  in  ordinary  routine 
water  examinations.  Indeed,  this  is  the  position  we 
have  maintained  in  both  our  earlier  editions. 

Chapter  V,  dealing  with  the  isolation  of  specific 
pathogenes  from  water,  has  been  extensively  rewritten 
and  extended.  The  use  of  the  Jackson  bile  medium 
for  the  preliminary  enrichment  of  the  typhoid  bacillus 
has  become  general  since  1908  and  a  number  of  suc- 
cessful isolations  have  been  reported  by  its  use;  so  that 
this  procedure  promises  to  be  of  increasing  importance 
in  the  future. 

In  regard  to  the  isolation  and  identification  of 
bacilli  of  the  colon  group  we  feel  that  the  time  has  come 
for  a  change  from  the  usual  American  practice  of  the 
past.  The  five  standard  tests  for  "  typical  B.  coli  " 
established  by  the  Committee  on  Standard  Methods 
in  its  1905  report  have  come  to  seem  more  and  more 
illogical  and  unscientific  to  most  practical  water  bac- 
teriologists. The  conviction  has  grown  that  they  go 
either  too  far  or  not  far  enough.  For  waters,  in  the 


PREFACE  TO  THIED  EDITION  xi 

United  States,  at  least,  it  seems  clear  that  all  of  the 
lactose-fermenting  group  of  bacilli  are  significant  of 
pollution  from  human  or  animal  sources  when  present 
in  considerable  numbers.  The  1912  report  of  the 
Standard  Methods  Committee  apparently  takes  this 
view  in  one  place,  while  retaining  the  five  tests  in  another 
section.  We  have  felt  it  best  to  place  ourselves  fairly 
and  fully  in  line  with  the  view  that  the  whole  group 
of  lactose-fermenting  bacilli  is  significant  and  that 
the  lactose  bile  fermentation  test  is  a  sufficient  identi- 
fication of  the  colon  group  for  ordinary  sanitary  pur- 
poses. This  broad  definition  is  the  one  upon  which 
we  have  based  our  general  discussion  of  the  colon 
group  in  Chapters  VI  and  VII.  In  Chapter  VIII 
we  have  discussed  the  subdivisions  of  the  group  as 
worked  out  by  MacConkey  and  others  and  their  special 
significance  with  respect  to  recent  and  remote  pollu- 
tion as  suggested  by  the  researches  of  Houston  and 
Clemesha. 

The  growing  importance  of  the  application  of  bac- 
teriology to  the  sanitary  study  of  shellfish  has  led 
us  to  include  a  new  chapter  dealing  with  this  subject, 
based  largely  upon  the  recent  report  of  the  Committee 
of  the  Laboratory  Section  of  the  American  Public 
Health  Association. 

Throughout  the  book  we  have  resorted  freely  to 
the  use  of  tables  of  actual  data  for  the  illustration  of 
the  various  points  discussed,  believing  that  ample 
familiarity  with  practical  examples  furnishes  the  only 
sound  basis  for  judgment  in  sanitary  water  exam- 
ination. 


xii  PEEFACE  TO  THIKD  EDITION 

For  the  benefit  of  the  student  the  chapters  have 
been  sub-divided  into  sections  with  prominent  headings 
indicating  the  general  topics  under  discussion. 

Massachusetts  Institute  of  Technology, 
BOSTON,  Mass. 

College  of  the  City  of  New  York, 
NEW  YORK,  N.  Y. 

June  i,  1913. 


TABLE  OF  CONTENTS 


CHAPTER  I 

PAGE 

THE  BACTERIA  IN  NATURAL  WATERS i 


CHAPTER  II 

THE  QUANTITATIVE  BACTERIOLOGICAL  EXAMINATION  or  WATER.  .     29 

CHAPTER  III 

THE   INTERPRETATION  OF  THE  QUANTITATIVE   BACTERIOLOGICAL 
EXAMINATION 51 

CHAPTER  IV 

DETERMINATION  OF  THE  NUMBER  OF  ORGANISMS  DEVELOPING  AT 
THE  BODY  TEMPERATURE 61 

CHAPTER  V 
THE  ISOLATION  OF  SPECIFIC  PATHOGENES  FROM  WATER 74 

CHAPTER  VI 

THE  COLON  GROUP  OF  BACILLI  AND  METHODS  FOR  THEIR  ISOLATION    99 

xiii 


xiv  TABLE  OF  CONTENTS 


CHAPTER  VII 

PAGE 

SIGNIFICANCE  or  THE  PRESENCE  OF  THE  COLON  GROUP  IN  WATER.   140 


CHAPTER  VIII 

VARIETIES  OF  COLON  BACILLI  AND  THEIR  SPECIAL  SIGNIFICANCE.  174 

CHAPTER  IX 
OTHER  INTESTINAL  BACTERIA 201 

CHAPTER  X 

THE  SIGNIFICANCE  AND  APPLICABILITY  OF  THE  BACTERIOLOGICAL 
EXAMINATION 215 

CHAPTER  XI 

BACTERIOLOGY  OF  SEWAGE  AND  SEWAGE  EFFLUENTS 228 

CHAPTER  XII 

BACTERIOLOGICAL  EXAMINATION  OF  SHELLFISH 244 

APPENDIX 265 


ELEMENTS  OF  WATER  BACTERIOLOGY 


CHAPTER  I 
THE  BACTERIA  IN  NATURAL  WATERS 

Bacteria  and  Their  Nutritive  Relations.  Bacteria  are 
the  most  numerous  and  the  most  widely  distributed 
of  living  things.  They  are  present  not  merely  at  the 
surface  of  the  earth  or  in  the  bodies  of  water  which 
partially  cover  it,  as  is  the  case  with  most  other  living 
things,  but  in  the  soil  itself,  and  in  the  air  above,  and 
in  the  waters  under  the  earth. 

Probably  no  organisms  are  more  sensitive  to  external 
conditions,  and  none  respond  more  quickly  to  slight 
changes  in  their  environment.  Temperature,  moisture, 
and  oxygen  are  of  importance  in  controlling  their  distri- 
bution; but  the  most  significant  factor  is  the  amount 
of  food  supply.  Bacteria  and  decomposing  organic 
matter  are  always  associated,  and  for  this  reason  a 
brief  consideration  of  the  general  relation  of  bacteria 
to  their  sources  of  food  supply  must  precede  the  study 
of  their  distribution  in  any  special  medium. 

The  bacteria  possess  greater  constructive  ability 
than  any  animal  organisms.  They  lack,  however, 


2  ELEMENTS  OF  WATER  BACTERIOLOGY 

the  power  of  green  plants  to  build  up  their  own  food 
from  compounds  like  carbon  dioxide  and  nitrates  which 
have  no  stored  potential  energy.  The  food  require- 
ments of  various  bacterial  types  differ,  however,  widely 
among  themselves.  Fischer  (1900)  has  divided  the 
whole  group  into  three  great  subdivisions  according 
to  the  nature  of  their  metabolism.  The  Proto trophic 
forms  are  characterized  by  minimal  nutrient  require- 
ments, including  organisms  like  the  nitrifying  bacteria 
which  require  no  organic  compounds  at  all,  but  derive 
their  nourishment  from  carbon  dioxide  or  carbonates, 
nitrites  and  phosphates,  or  from  inorganic  ammonium 
salts.  A  second  group  of  Metatrophic  bacteria  includes 
those  forms  which  require  organic  matter,  nitrogenous 
and  carbonaceous,  but  are  not  dependent  on  the  fluids 
of  the  living  plant  or  animal.  Finally,  the  Para  trophic 
bacteria  are  the  true  parasites,  which  exist  only  within 
the  living  tissues  of  other  organisms.  These  sub- 
divisions, like  all  groups  among  the  lower  plants,  are 
not  sharply  defined,  and  the  metatrophic  bacteria  in 
particular  exhibit  every  gradation,  from  types  which 
grow  in  water  with  a  trace  of  free  ammonia  to  organisms 
like  the  colon  bacillus  which  normally  occur  on  the 
surface  of  the  plant  or  animal  body,  feeding  upon  the 
fluids  or  on  the  extraneous  material  collected  upon  its 
surface. 

The  vast  majority  of  bacteria  belong  to  the  second, 
or  metatrophic  group,  living  as  saprophytes  on  dead 
organic  matter  wherever  it  may  occur  in  nature,  and 
particularly  in  that  diffuse  layer  of  decomposing  plant 
and  animal  material  which  we  call  the  humus,  or  surface 


THE  BACTERIA  IN  NATURAL  WATERS  3 

layer  of  the  soil.  Wherever  there  is  life,  waste  matter 
is  constantly  being  produced,  and  this  finds  its  way 
to  the  earth  or  to  some  body  of  water.  The  excretions 
of  animals,  the  dead  tissues  and  broken-down  cells  of 
both  animals  and  plants,  as  well  as  the  wastes  of  domestic 
and  industrial  life,  all  eventually  find  their  way  to  the 
soil.  In  a  majority  of  cases  these  substances  are  not 
of  such  chemical  composition  that  they  can  be  utilized 
at  once  by  green  plants  as  food,  but  it  is  first  necessary 
that  they  go  through  a  decomposition  or  transforma- 
tion in  which  their  chemical  nature  becomes  changed; 
and  it  is  as  the  agents  of  this  transformation  that 
bacteria  assume  their  greatest  importance  in  the  world 
of  life. 

We  may  take  the  decomposition  of  a  comparatively 
simple  excretory  product,  urea,  as  an  example  of  the 
part  which  the  bacteria  play  in  the  preparation  of 
plant  food.  Through  the  activity  of  an  enzyme  pro- 
duced by  certain  bacteria  this  compound  unites  with 
two  molecules  of  water  and  is  converted  into  ammonium 
carbonate, 


+  2H20  =  (NH4)2C03. 
NH2 


This,  however,  is  only  part  of  the  process.  While 
green  plants  can  derive  their  necessary  nitrogen  in  part, 
at  least,  from  ammonium  compounds  it  is  a  well- 
established  fact  that  this  element  is  often  obtained 
more  readily  from  nitrates,  and  there  are  other  bacteria 
which  as  a  further  step  oxidize  the  ammoniacal  nitro- 


4  ELEMENTS  OF  WATEE  BACTERIOLOGY 

gen  to  a  more  available  form.  This  process  of  oxida- 
tion is  known  as  nitrification,  and  takes  place  in  a  suc- 
cession of  steps,  the  organic  nitrogen  being  first  con- 
verted to  the  form  of  ammonium  salts,  and  these  in 
turn  to  nitrites  and  nitrates,  the  oxygen  used  coming 
from  the  air.  Several  groups  of  organisms  are  instru- 
mental in  bringing  about  this  conversion.  It  is  gen- 
erally assumed  that  one  group  attacks  the  ammonium 
compounds  and  changes  them  to  nitrites;  while  another 
group  completes  the  oxidation  to  nitrates.  In  the 
latter  form  nitrogen  is  readily  taken  up  by  green  plants 
to  be  built  up  into  more  complex  albuminoid  sub- 
stances (organic  nitrogen)  through  the  constructive 
power  of  chlorophyll. 

This  never-ending  cycle  is  illustrated  in  the  accom- 
panying figure,  devised  by  Sedgwick  (Sedgwick,  1889)  to 
illustrate  the  transformations  of  organic  nitrogen  in 
nature,  the  increasing  size  and  closeness  of  the  spiral 
on  the  left-hand  side  indicating  the  progressive  com- 
plexity of  organic  matter  as  built  up  by  the  chlorophyll 
bodies  of  green  plants  in  the  sunlight,  and  the  other 
half  of  the  figure  the  reverse  process,  carried  out  largely 
by  the  bacteria.  In  nature  there  are  many  short 
circuits,  as,  for  instance,  when  dead  organic  matter 
is  used  as  food  for  animals  and  built  up  into  the  living 
state  again  without  being  nitrified  and  acted  upon 
by  green  plants;  but  the  complete  cycle  of  organic 
nitrogen  is  as  indicated  on  the  diagram. 

We  have  dwelt  thus  at  length  upon  the  general 
relation  between  bacteria  and  organic  decomposition 
because  in  this  relation  will  be  found  the  master  key 


THE  BACTEEIA  IN  NATURAL  WATERS  5 

to  the  distribution  of  bacteria  in  water  as  well  as  in 
other  natural  habitats.  It  is  true  that  certain  peculiar 
forms  may  at  times  multiply  in  fairly  pure  waters;  but, 
in  general,  large  numbers  of  bacteria  are  found  only  in 
connection  with  the  organic  matter  upon  which  they 
feed.  Such  organic  matter  is  particularly  abundant 
in  the  surface  layer  of  the  soil.  Here,  therefore,  the 
bacteria  are  most  numerous;  and  in  other  media  their 


THE  SPHERE 

OF 
ORGANISMS 

AND 
THE  HISTORY 

OF 
ORGANIC  MATTER, 


numbers  vary  according  to  the  extent  of  contact  with 
the  living  earth. 

Classification  of  Waters.  Natural  waters,  then, 
group  themselves  from  a  bacteriological  standpoint 
in  four  well-marked  classes,  according  to  their  relation 
to  the  rich  layers  of  bacterial  growth  upon  the  surface  of 
the  globe.  There  are  first  the  atmospheric  waters  which 
have  never  been  subject  to  contact  with  the  earth; 
second,  the  surface-waters  immediately  exposed  to  such 


6  ELEMENTS  OF  WATER  BACTERIOLOGY 

contamination  in  streams  and  pools;  third,  stored  waters, 
the  lakes  and  large  ponds  in  which  storage  has  reduced 
bacterial  numbers  to  a  state  of  comparative  purity; 
and  fourth,  the  ground-waters  from  which  previous 
contamination  has  been  even  more  completely  removed 
by  filtration  through  the  deeper  layers  of  the  soil. 

Bacterial  Content  of  Various  Waters.  Even  rain  and 
snow,  the  sources  of  our  potable  waters,  are  by  no 
means  free  from  germs,  but  contain  them  in  numbers 
varying  according  to  the  amount  of  dust  present  in 
the  air  at  the  time  of  the  precipitation.  After  a  long- 
continued  storm  the  atmosphere  is  washed  nearly  free 
of  bacteria,  so  that  a  considerable  series  of  sterile  plates 
may  often  be  obtained  by  plating  i-c.c.  samples.  These 
results  are  in  harmony  with  the  observations  of  Tissandier 
(reported  by  Duclaux,  1897),  wno  found  that  the  dust 
in  the  air  amounted  to  23  mg.  per  cubic  meter  in  Paris 
and  4  mg.  in  the  open  country.  After  a  rainfall  these 
figures  were  reduced  to  6  mg.  and  0.25  mg.,  respectively. 

With  regard  to  what  may  be  considered  normal  values 
for  rain  it  is  difficult  to  give  satisfactory  figures.  Those 
obtained  by  Miquel  (Miquel,  1886)  during  the  period 
1883-1886  showed  on  the  average  4.3  bacteria  per  c.c. 
in  the  country  (Montsouris)  and  19  per  c.c.  in  Paris. 
Snow  shows  rather  higher  numbers  than  rain.  Janowski 
(Janowski,  1888)  found  in  freshly  fallen  snow  from  34 
to  463  bacteria  per  c.c.  of  snow-water. 

As  soon  as  the  rain-drop  touches  the  surface  of  the 
earth  its  real  bacterial  contamination  begins.  Rivulets 
from  ploughed  land  or  roadways  may  often  contain 
several  hundred  thousand  bacteria  to  the  cubic  centi- 


THE  BACTERIA  IN  NATURAL  WATERS  7 

meter;  and  furthermore  the  amounts  of  organic  and 
mineral  matters  which  serve  as  food  materials,  and  thus 
become  a  factor  in  later  multiplication  of  organisms, 
are  greatly  increased. 

In  the  larger  streams  several  conditions  combine  to 
make  these  enormous  bacterial  numbers  somewhat 
lower.  Ground-water  containing  little  microbic  life 
enters  as  a  diluting  factor  from  below.  The  larger 
particles  of  organic  matter  are  removed  from  the  flow- 
ing water  by  sedimentation;  many  earth  bacteria, 
for  which  water  is  an  unfavorable  medium,  gradually 
perish;  and  in  general  a  new  condition  of  equilibrium 
tends  to  be  established.  It  is  difficult,  however,  to 
find  a  river  in  inhabited  regions  which  does  not  con- 
tain several  hundreds  or  thousands  of  bacteria  to  the 
cubic  centimeter.  Furthermore,  heavy  rains  which 
introduce  wash  from  the  surrounding  watershed  may 
at  any  time  upset  whatever  equilibrium  exists,  and 
surface-waters  are  apt  to  show  sudden  fluctuations 
in  their  bacterial  content. 

Seasonal  Variation  of  Bacteria  in  Surface  Waters. 
Sharp  variations  in  bacterial  content  are  particularly 
apt  to  occur  in  the  spring  and  fall  as  a  result  of  the 
rain  and  melting  snow  at  those  seasons.  The  high 
numbers  shown  for  various  rivers  in  the  table  on  page 
8  illustrate  this  point. 

The  rainfall  is  the  main  factor  which  causes  these  sea- 
sonal variations;  but  its  specific  effect  differs  with  dif- 
ferent streams.  The  immediate  result  of  a  smart  shower 
is  always  to  increase  contamination  by  introducing  fresh 
wash  from  the  surface  of  the  ground.  More  prolonged 


s 


ELEMENTS  OF  WATER  BACTERIOLOGY 


SEASONAL  VARIATIONS  IN  BACTERIAL  CONTENT  OF 
RIVER  WATERS.  BACTERIA  PER  C.C.  MONTHLY 
AVERAGE 


River. 

Year. 

Jan. 

Feb. 

Mar. 

April. 

May. 

June. 

Thames  x  

1905-6 

2075 

1,679 

1,161 

277 

I  064 

8? 

Lea1  

1905-6 

5,192 

3,083 

1,308 

471 

1,350 

598 

New  1  

1905-6 

I  455 

I  304 

291 

I4.Q 

-?  cr  2 

108 

Mississippi  2  .  .  .  . 

1900-01 

972 

2,871 

i,795 

3,597 

2,152 

2,007 

Potomac  3  

1906-7 

4,400 

I,OOO 

11,500 

3,700 

7  co 

77  300 

Merrimac  4  

14,200 

14.800 

10,300 

3,600 

1,900 

9,600 

Susquehanna  5.  . 

1906 

9,510 

21,228 

31,326 

39,905 

6,187 

2,903 

River. 

Year. 

July. 

/  ug. 

Sept. 

Oct. 

Nov. 

Dec. 

Thames  1  

190=5—6 

952 

I  633 

74-O 

Lea1  

1905-6 

1,190 

7,946 

2.CKO 

New1  

1905-6 

450 

718 

621 

Mississippi  2  .  .  .  . 

1900-01 

1,832 

805 

2,021 

Potomac  3  

1906-7 

2,700 

3,000 

6,2OO 

2,300 

1,  800 

6,9OO 

Merrimac  4  

1905 

3,900 

19,500 

I3-500 

39,800 

8,700 

Susquehanna  $    . 

1906 

685 

1,637 

836 

7,575 

26,224 

37,525 

1  Houston,  19060,  19066.  4  Massachusetts,  1906. 

2  New  Orleans,  1903.  5  Harrisburg,  1907. 

3  Figures  obtained  through  courtesy  of  F.  F.  Longley. 

moderate  rain,  however,  exerts  an  opposite  effect,  and 
after  the  main  impurities  which  can  be  washed  away 
have  been  removed,  may  dilute  the  stream  with  water 
purer  than  itself.  What  the  net  effect  of  rain  may  be 
depends,  therefore,  on  the  character  of  the  stream. 
A  river  of  fairly  good  quality  shows  its  highest  numbers 
in  rainy  periods.  With  a  highly  polluted  stream,  on 
the  other  hand,  the  constant  influx  of  sewage  over- 
balances occasional  contributions  of  surface  contamina- 
tion. Thus  Gage  (1906)  shows  in  the  following  table 
that  the  bacterial  content  of  the  Merrimac  is  highest 
when  the  stream  is  lowest,  that  is,  when  its  sewage 
content  is  least  subject  to  dilution. 


THE  BACTERIA  IN  NATURAL  WATERS 


VARIATIONS  IN  BACTERIAL  CONTENT,  MERRIMAC  RIVER 
GAGE  (1906) 


Flow  of  St-eam.     Cubic 

Bacteria 

per  c.c. 

B.  coli 

per  c.c. 

Square  Mile  of  Watershed 

Canal. 

ntake. 

Canal. 

Intake. 

Less  than  i  

7,500 

10,800 

66 

.88 

1—2 

6  800 

6  200 

CQ 

qi 

2-4         

3,600 

5,6oo 

2O 

2Q 

Over  4 

3  AOO 

2   IOO 

16 

2O 

The  contrast  between  the  two  classes  of  rivers  is  well 
brought  out  in  a  study  of  the  Lahn  and  the  Wieseck, 
by  Kisskalt  (1906);  and  the  table  below,  compiled  from 
his  data,  gives  an  excellent  idea  of  the  total  numbers  of 
bacteria  and  their  seasonal  fluctuations  in  a  stream  of 
fair  quality  (the  Lahn)  and  a  highly  polluted  one  (the 
Wieseck).  In  the  former  case  the  bacterial  numbers 
are  highest  when  rain  brings  surface  pollution;  in  the  lat- 
ter, when  the  sewage  constantly  present  is  least  diluted. 

MONTHLY  VARIATIONS  OF  BACTERIA  IN  A  NORMAL  AND 
POLLUTED  STREAM 

KISSKALT,  1906 


Date. 

Bacteria 

per  c.c. 

Date. 

Bacteria 

per  c.c. 

1904. 

Lahn. 

Wieseck. 

1904-5- 

Lahn. 

Wieseck. 

My 

3l8 

104  ooo 

December  * 

I  22O 

21  2OO 

July  
August  
October  l  .  .  . 
October  l  .  .  . 

132 

840 

1,235 
420 

156,800 

98,400 
28,400 
58,000 

January  l  .  . 
February  1  . 
March1.... 
April  !  

3,668 
5,38o 

I,2IO 
4,025 

29,920 
1  1  ,9OO 
8,250 
5,QIO 

November 

2  34O 

7Q    2OO 

May 

C7o 

14  800 

November  l  . 

I,74O 

5  2  ,000 

June  .  , 

686 

^o  180 

December  l  .  . 

780 

28,600 

1  Rain  or  high  water  due  to  previous  thaw. 


10 


ELEMENTS  OF  WATER  BACTERIOLOGY 


Effect  of  Storage  upon  Bacteria  in  Water.  In  stand- 
ing waters  all  the  tendencies  which  make  for  the  reduc- 
tion of  bacteria  are  intensified,  and  when  a  river 
passes  into  a  natural  or  artificial  reservoir  a  notable 
reduction  in  numbers  occurs.  The  table  below  shows 
the  striking  effect  produced  upon  the  water  of  the 
Potomac  River  by  its  successive  passage  through  the 
three  reservoirs  of  the  Washington  water  supply  in 
the  first  nine  months  of  1907.  We  owe  these  figures 
to  the  courtesy  of  Mr.  F.  F.  Longley,  the  engineer  then 
in  charge  of  the  Washington  filter  plant. 

REDUCTION  OF  BACTERIA  IN  WASHINGTON  RESERVOIRS. 
BACTERIA  PER  C.C.,  MONTHLY  AVERAGE,  1907 


Potomac 
River. 

Dalecarlia 
Reservoir. 

Georgetown 
Reservoir. 

Washington 
City  Reservoir. 

January  
February  .... 
March  . 

4,400 
I,OOO 
II  500 

2,400 

95° 
8  300 

2,2OO 
I,OOO 
7  2OO 

950 

750 
•2  600 

April  

3.700 

2  IOO 

I  4OO 

47  ^ 

M!ay 

7  ^O 

•7  tro 

•2  2  C 

June  
July  .  . 

2,300 

2  7OO 

950 
600 

600 
3^O 

IOO 
1  60 

August  
September  .  .  . 

3,000 
6.  200 

275 

425 
I.QOO 

80 

230 

The  still  more  striking  results  obtained  at  London 
are  indicated  in  the  table  on  page  n. 

When  the  water  which  enters  a  pond  or  a  reservoir 
has  already  undergone  considerable  storage  and  reached 
a  comparatively  stable  condition,  the  diminution  due  to 
additional  storage  may  be  almost  negligible.  Thus 
Philbrick  (1905)  found  that  the  influent  water  of  the 
Chestnut  Hill  Reservoir  of  the  Metropolitan  Water 


THE  BACTEEIA  IN  NATURAL  WATERS 


11 


Works  of  Boston  contained  on  the  average  during  the 
eleven  years,  1893-1903,  220  bacteria  per  c.c.,  and  the 
effluent  179.  In  many  individual  months,  and  in  some 
whole  years,  the  effluent  contained  more  than  the 
influent. 

AVERAGE    REDUCTION    OF    BACTERIA    BY    STORAGE    AT 
LONDON 

(HOUSTON,  1909) 


Water 

Storage, 
Days. 

Bacteria  per  c.c. 

Gelatin 
20°. 

Agar 
37°. 

Bile-salt 
Agar  37°. 

Raw  Thames  River      

4405 
175 
208 
362 
8135 
67 

280 
34 
44 
52 
382 
II 

41 

2 

5 
8 

34 

i 

Do.  stored  at  Staines  

95 
15 
14 

"5s" 

Do  stored  at  Chelsea 

Do.  stored  at  Lambeth  
Raw  Lee  River  

Do.  stored  

The  seasonal  variations  in  the  bacterial  content  of  a 
large  pond  or  lake  follow  a  somewhat  different  course 
from  those  observed  in  a  stream.  Philbrick,  in ,  the 
paper  just  cited,  gives  the  figures  tabulated  below  for 
the  Chestnut  Hill  Reservoir  of  the  Metropolitan  Water 
Works  (Boston).  The  averages  are  based  on  weekly 
analyses  covering  the  eleven  years,  1893-1903. 

MONTHLY    VARIATIONS    IN    BACTERIAL    CONTENT    OF 
CHESTNUT  HILL  RESERVOIR,    1893-1903 


Month. 

J. 

F. 

M. 

A. 

M. 

J. 

J. 

A. 

S. 

0.    |    K. 

D. 

Bacteria 

per  c.c. 

82 

73 

7i 

123 

69 

73 

82 

95 

134 

89 

103 

96 

12 


ELEMENTS  OF  WATER  BACTERIOLOGY 


The  marked  increase  in  April  and  September  is  the 
notable  feature  of  these  analyses;  and  this  is  due  to  the 
effect  of  the  spring  and  fall  overturns  which,  in  the 
months  in  question,  stir  up  the  decomposing  organic 
matter  at  the  bottom  and  distribute  it  through  the 
reservoir.  The  slight,  but  steady,  increase  during  the 
warm  months  from  May  to  August  is  also  probably 
significant. 

On  the  whole  it  may  be  said  that  the  bacterial  content 
of  a  lake  or  pond  should  not  be  more  than  one  or  two 
hundred  per  c.c.  and  may  often  be  under  a  hundred. 
The  student  will  find  numerous  analyses  of  natural 
waters  in  Frankland's  classic  work  (Frankland,  1894). 
He  notes,  for  example,  that  the  Lake  of  Lucerne  con- 
tained 8  to  51  bacteria  per  c.c.,  Loch  Katrine  74,  and 
the  Loch  of  Lintralthen  an  average  of  170.  The  water 
of  Lake  Champlain  examined  by  one  of  us  (S.  C.  P.)  in 
1896  contained  on  an  average  82  bacteria  per  c.c.  at  a 
point  more  than  two  miles  out  from  the  city  of  Burling- 
ton. Certain  surface  water-supplies  near  Boston  studied 
by  Nibecker  and  one  of  us  (Winslow  and  Nibecker,  1903), 
gave  the  following  results: 


City. 

Number  of 
Samp  es. 

Average  Number 
of  Bacteria  per  c.c. 

Wakefield  

7 

CO 

Lynn 

6 

16 

Plymouth  

6 

•2  r 

Cambridge  

c 

04 

Salem 

c 

222 

Medford                  .    . 

C 

C.24. 

Taunton  

4 

13 

Peabody 

? 

I4.I 

THE  BACTEEIA  IN  NATURAL  WATERS 


13 


In  sea-water,  too,  bacterial  numbers  are  small,  as 
noted  by  Russell  at  Naples  (Russell,  1891)  and  Wood's 
Hole  (Russell,  1892),  and  in  salt  as  in  fresh  water  the 
amount  of  bacterial  life  decreases  in  general  as  one 
passes  downward  from  the  surface  and  outward  from 
the  shore.  Otto  and  Neumann  (1904)  obtained  the 
results  summarized  below  at  various  points  on  the 
high  seas  between  Portugal  and  Brazil.  Near  the 
European  coast  numbers  were  much  higher. 

BACTERIA     IN     THE     ATLANTIC    OCEAN.       (OTTO    AND 
NEUMANN,  1904.)     BACTERIA  PER  C.C. 


Nearest  Land. 

Depth  ir 

Meters. 

5 

50 

IOO 

2OO 

Canary  Islands  

1  20 

76 

2O 

I 

Cape  Verde  Islands  

58 

16 

64 

6 

St  Paul  Island 

20 

480 

54 

4 

Pernambuco  

48 

168 

83 

14 

Drew  (1912)  finds  high  numbers  of  bacteria  in  surface 
sea- water  off  the  Bahamas,  ranging  from  13,000  to 
16,000,  falling  off  below  200  fathoms  (in  the  cold  bottom 
waters  at  10°  C.  or  below)  to  o  to  17. 

Factors  Influencing  the  Diminution  of  Bacteria  in 
Surface-waters.  The  decrease  in  numbers  which  takes 
place  when  a  surface-water  is  stored  in  a  pond  or  reservoir 
indicates  that  the  forces  which  tend  to  produce  bacterial 
self-purification  are  important  ones.  It  is  necessary 
to  consider  in  somewhat  more  detail  just  what  these 
forces  are,  in  order  to  gauge  their  potency  in  any 
particular  instance. 


14         ELEMENTS  OF  WATER  BACTERIOLOGY 

Chief  of  them  appear  to  be  sedimentation,  the  activ- 
ity of  other  micro-organisms,  light,  temperature,  and 
food-supply,  and  perhaps  more  obscure  conditions  such 
as  osmotic  pressure. 

The  subsidence  of  bacteria,  either  by  virtue  of  their 
own  specific  gravity,  or  as  the  result  of  their  attachment 
to  particles  of  suspended  matter,  is  unquestionably 
partly,  if  not  largely,  responsible  for  changes  in  the 
number  of  bacteria  in  the  upper  layers  of  water  at  rest 
or  in  very  sluggish  streams.  The  results  of  numerous 
investigations  by  different  workers  seem  to  indicate  that 
sedimentation  of  the  bacteria  themselves  takes  place 
slowly,  and  that  the  difference  in  numbers  between 
the  top  layer  and  the  bottom  layer  of  water  in  tall 
jars  in  laboratory  experiments  of  a  few  days'  duration 
is  very  slight  or  quite  within  the  limits  of  experimental 
error  (Tiemann  and  Gartner,  1889).  Different  species 
may,  of  course,  be  differently  affected  (Scheurlen, 
1891).  It  must  be  remembered,  however,  that  in 
natural  streams  bacteria  are  to  a  great  extent  attached 
to  larger  solid  particles  upon  which  the  action  of  gravity 
is  more  important.  Spitta  (1903)  found  that  from  one- 
fifth  to  one-half  of  the  bacteria  in  canal  water  may 
be  attached  to  gross  particles,  as  evidenced  by  their 
sedimentation  in  a  few  hours.  Jordan  (Jordan,  1900) 
is  firmly  of  the  opinion  that  in  the  lower  part  of  the 
Illinois  River,  where  there  is  a  fall  of  but  30  feet  in 
225  miles,  the  influences  summed  up  by  the  term 
sedimentation  are  sufficiently  powerful  to  obviate  the 
necessity  for  summoning  another  cause  "  to  explain 
the  diminution  n\  numbers  of  bacteria,"  and  he  further 


THE  BACTERIA  IN  NATURAL  WATERS  15 

adds:  "  It  is  noteworthy  that  all  the  instances  recorded 
in  the  literature  where  a  marked  bacterial  purification 
has  been  observed  are  precisely  those  where  the  con- 
ditions have  been  most  favorable  for  sedimentation." 

Little  is  known  as  to  the  share  of  other  organisms  in 
hastening  the  decrease  of  bacteria  in  stored  water. 
Doubtless  predatory  Protozoa  play  some  part  in  the 
process.  Huntemiiller  (1905)  after  infecting  water 
containing  flagellate  Protozoa  with  typhoid  bacilli, 
found  the  Protozoa  crowded  with  bacteria;  and  he 
observed  under  the  microscope  the  actual  ingestion 
of  the  living  and  motile  bacilli.  Korschun  (1907)  and 
others  have  obtained  similar  results  and  consider  the 
activity  of  Protozoa  to  be  an  important  factor  in  self- 
purification.  Fehrs  (1906)  found  that  typhoid  bacilli 
would  live  for  7  days  in  unsterilized  Goltingen  tap  water, 
for  46  days  in  the  same  water  sterilized,  and  for  13 
days  in  water  inoculated  with  a  culture  of  flagellate 
Protozoa  after  sterilization.  Water  bacteria  were  of 
course  added  with  the  Protozoa.  Stokvis  and  Swel- 
lengrebel  (1911)  have  shown  that  ciliated  infusoria 
may  also  consume  considerable  quantities  of  bacteria 
under  favorable  conditions  as  to  oxygen  and  temperature, 
and  Horhammer  (1911)  reports  that  certain  Crustacea 
such  as  Cyclops  may  devour  considerable  quantities 
of  typhoid  bacilli  when  present  in  masses  from  cultures? 
stained  with  methylene  blue,  and  suspended  in  water. 

Certain  bacteriologists  have  held  that  the  toxic  waste 
products  of  the  bacteria  themselves  may  render  water 
unfit  for  their  own  development.  Horrocks  (Horrocks, 
1901),  Garre  (Garre,  1887),  Zagari  (Zagari,  1887)  and 


16         ELEMENTS  OF  WATER  BACTERIOLOGY 

Freudenreich  (Freudenreich,  1888)  have  shown  that  an 
"  antagonism "    exists    when    bacteria    are    grown    in 
artificial  culture  media,  such  that  the  substratum  which 
has  supported  the  growth  of  one  form  may  be  rendered 
antiseptic  to  another.     Frost   (1904)   has  exhaustively 
studied   the  phenomenon   of   antagonism  by   exposing 
typhoid  bacilli  in  collodion  sacs  to  the  action  of  certain 
soil   and   water   bacteria   growing  in  broth.     Artificial 
culture  media,  however,  offer  conditions  for  bacterial 
development  which  are  scarcely  paralleled  in  natural 
waters.     It  is  difficult  to  believe  that  under  ordinary 
conditions  poisons  are  produced  of  such  power  as  to 
render  a  stream  or  lake  specifically  toxic  for  any  par- 
ticular type  of  bacteria.     It  does   appear   indeed  from 
the  experiments  of  Jordan,  Russell  and  Zeit  (1904),  and 
Russell  and  Fuller  (1906),  which  will  shortly  be  referred 
to  more  fully,  that  the  life  of  typhoid  germs  is  shorter 
in  water  containing  large  numbers  of  other  bacteria 
than  in  that  of  greater  purity.     Horrocks  (1899),  too, 
found   freshly   isolated    typhoid   bacilli   alive  in  sterile 
sewage  after  60  days;   while  they  disappeared  in  5  days 
when  B.  coli  was  also  present.     These  phenomena  may 
be  due,  however,  to  a  struggle  for  oxygen,  or  for  food, 
rather  than  to  the  assumed  presence  of  highly  toxic 
bacterial  products,  of  which  there  is  no   independent 
evidence. 

Many  investigations  conducted  since  the  pioneer 
researches  of  Downes  and  Blunt  (Downes  and  Blunt, 
1877)  have  confirmed  the  results  reported  by  them, 
which  showed  that  direct  sunlight  is  fatal  to  most 
bacteria  in  the  vegetative  state  and  even  to  spores  if 


THE  BACTEEIA  IN  NATURAL  WATERS          17 

the  exposure  be  sufficiently  long,  while  diffused  light 
is  harmful  in  a  less  degree.  Opinions  vary  as  to  the 
degree  to  which  light  is  active  in  destroying  the  bacteria 
in  natural  waters.  Buchner  (Buchner,  1893)  found 
by  experiment  that  the  bactericidal  power  of  light 
extends  to  a  depth  of  about  three  meters  before  it 
becomes  imperceptible.  On  the  other  hand,  Procaccini 
(Procaccini,  1893)  found  that  when  sunlight  was  passed 
vertically  through  60  cm.  of  drain- water  the  lower 
layers  contained  nearly  as  many  bacteria  after  3  hours' 
treatment  as  before  the  exposure.  The  middle  and 
upper  portions  showed  a  great  falling  off  in  numbers, 
however. 

But  few  studies  have  been  made  of  the  effect  of  light 
on  bacteria  in  flowing  water.  Jordan  (Jordan,  1900) 
has  investigated  several  Illinois  streams  and  arrived 
at  the  conclusion  that  in  moderately  turbid  water,  at 
least,  the  sun's  rays  are  virtually  without  action.  On 
the  other  hand,  Rapp  has  observed  a  considerable 
reduction  of  the  bacteria  in  the  Isar  at  Pullach  after  the 
period  of  diurnal  insolation,  as  shown  by  the  table  on 
the  following  page.  Clemesha  (191 2a)  attributes  very 
great  importance  to  the  action  of  light  in  the  self- 
purification  which  takes  place  in  Indian  lakes  and 
rivers;  his  opinion  is  apparently  not  based  on  com- 
parative experiments  including  and  excluding  this 
factor,  but  chiefly  on  the  greater  numbers  of  intestinal 
bacteria  at  the  bottom  as  compared  with  the  superficial 
layers  of  water. 

It  is  unnecessary  to  dwell  in  detail  upon  the  effect 
which  the  lack  of  nutritive  elements  must  exert  upon 


18 


ELEMENTS  OF  WATER  BACTERIOLOGY 


intestinal  bacteria  and  soil  bacteria  in  waters  of  ordinary 
purity.  Comparative  studies  of  culture  media,  to  be 
quoted  in  the  succeeding  chapter,  will  show  how  del- 
icately the  bacteria  respond  to  comparatively  slight 
changes  in  their  food-supply.  Wheeler  (1906)  found 
that  typhoid  bacilli  would  persist  in  almost  undimin- 
ished  numbers  in  sterilized  water  from  a  polluted  well 
containing  considerable  organic  matter  and  kept  in 
the  dark  at  20  degrees,  while  in  purer  water  or  in  the 
light  they  died  out  in  from  2  to  6  weeks. 

EXAMINATIONS  OF  THE  ISAR  AT  PULLACH 

(RAPP,  1903) 
(A}  Carried  out  September  26.  i8p8,  no  rain  having  fallen  for  three  weeks 


Temperature. 

Time  o    the 
Experiment. 

Bacteria  per  c.c. 

of  the  Water. 

of  the  Air. 

i3.o°C. 

8Q  O    /"^ 
.  0       C. 

7.3op.M. 

146 

I2.I°C. 

7.o°C. 

9.30P.M. 

270 

10.  5°  C. 

6.2°C. 

5.00  A.M. 

370 

10.2°  C. 

8.2°C. 

8.00  A.M. 

320 

(B)    Carried  out  November  28,  1898,  no  rain  having  fallen  for  some  time 


5-5°  C. 

3-o°C. 

6.OO  P.M. 

266 

5-5°C. 

2-5°C. 

8.00P.M. 

402 

5.5°C. 

2.0°C. 

10.  00  P.M. 

482 

5.o°C. 

2.0°C. 

3-OO  A.M. 

532 

4.5°C. 

2-5°C. 

7.30A.M. 

400 

Whipple  and  Mayer  (1906)  have  called  attention  to 
another  important  factor  in  the  general  problem.  They 
find  that  the  presence  of  oxygen  is  essential  to  the  per- 


THE  BACTERIA  IN  NATURAL  WATERS 


19 


sistence  of  typhoid  and  colon  bacilli  in  water,  although 
in  nutrient  media  both  forms  may  thrive  under  anaerobic 
conditions. 


EFFECT  OF  OXYGEN  ON  VIABILITY  OF  TYPHOID  BACILLI 
IN   STERILE   TAP   WATER 

WHTPPLE  AND  MAYER,  1906 


Tubes  Kept  in  Air. 

Tubes  Kept  in  Hydrogen. 

Period  in  Days 

Bacteria 
per  c.c. 

Per  Cent. 

Bacteria 
per  c.c. 

Per  Cent. 

o 

600,000 

IOO.O 

6oo,000 

IOO.O 

2 

455;°°° 

76.0 

2,400 

0.4 

4 

190,000 

32.0 

25 

0.004 

8 

120,000 

20.  O 

O 

O.O 

12 

67,000 

II.  0 

0 

O.O 

18 

25,000 

4-2 

o 

O.O 

26 

9,250 

i-5 

o 

O.O 

33 

2,150 

0.6 

0 

0.0 

40 

132 

O.O2 

0 

O.O 

47 

6 

O.OOI 

o 

O.O 

54 

o 

o.ooo 

0 

0.0 

Various  inorganic  constituents  of  the  medium  undoubt- 
edly exercise  an  important  influence  upon  the  life  of 
bacteria  in  water;  and  the  mutual  interaction  of  the 
different  substances  present  is  a  highly  complex  one. 
Thus  Winslow  and  Lochridge  (1906)  report  that  five 
parts  of  dissociated  hydrogen  per  million  parts  of  tap 
water  (0.005  normal  HC1)  is  fatal  to  typhoid  bacilli, 
while  ten  times  as  much  acid  is  required  for  sterilization 
when  i  per  cent  of  peptone  is  present  to  check  the 
dissociation  of  the  hydrogen.  In  Hazen  and  Whipple's 
study  of  the  Allegheny,  Monongahela  and  Ohio  rivers 


20          ELEMENTS  OF  WATER  BACTERIOLOGY 

at  Pittsburgh  the  antiseptic  effect  of  acid  wastes  was 
strikingly  shown.     (Engineering  News,  1912.) 

Although  it  is  hard  to  estimate  the  exact  importance 
of  each  factor,  the  general  phenomena  of  the  self- 
purification  of  streams  are  easy  to  comprehend.  A 
small  brook,  immediately  after  the  entrance  of  polluting 
material  from  the  surface  of  the  ground,  contains  many 
bacteria  from  a  diversity  of  sources.  Gradually  those 
organisms  adapted  to  life  in  the  earth  or  in  the  bodies 
of  plants  and  animals  die  out,  and  the  forms  for  which 
water  furnishes  ideal  conditions  survive  and  multiply. 
It  is  no  single  agent  which  brings  this  about,  but  that 
complex  of  little-understood  conditions  which  we  call 
the  environment.  If  any  one  thing  is  of  prime  impor- 
tance it  is  probably  the  food-supply,  for  only  certain 
bacteria  are  able  to  multiply  in  the  presence  of  the 
small  amount  of  organic  matter  present  in  ordinary 
potable  waters.  As  Jordan  (Jordan,  1900)  has  said: 
"  In  the  causes  connected  with  the  insufficiency  or 
unsuitability  of  the  food-supply  is  to  be  found,  I  believe, 
the  main  reason  for  the  bacterial  self-purification  of 
streams." 

Effect  of  Temperature  upon  Bacteria  in  Water. 
The  effect  of  temperature  upon  the  survival  of  bac- 
teria in  water  varies  according  to  this  primary  con- 
dition of  food-supply  which  has  just  been  discussed. 
When  bacteria  are  in  a  medium  in  which  they  are  able 
to  grow  and  multiply,  warmth,  within  reasonable  limits 
of  course,  favors  their  development,  At  times  this 
may  be  true  even  of  certain  intestinal  bacteria  in  water. 
Thus  at  Harrisburg,  Pa.,  a  series  of  B.  coli  examinations 


THE  BACTERIA  IN  NATURAL  WATERS          21 

made  in  the  midsummer  of  1906  showed  positive 
results  in  7  per  cent  of  the  samples  of  water  entering  the 
storage  reservoir  and  in  27  per  cent  of  the  samples 
leaving  it.  The  storage  period  in  this  case  was  about 
two  days  and  the  temperature  of  the  water  in  the 
reservoir  was  nearly  at  blood  heat  (Harrisburg,  1907). 
Clemesha  (1912*)  has  recently  made  an  exhaustive  study 
of  this  multiplication  of  coli-like  microbes  in  warm 
waters  and  has  shown  that  it  is  confined  to  certain 
particular  types  within  the  colon  group.  For  most 
intestinal  bacteria  the  conditions  necessary  for  growth 
and  multiplication  are  not  realized  in  water  and  an 
entirely  different  temperature  effect  is  manifest.  When 
a  bacterium  cannot  multiply,  the  only  vital  activity 
which  can  take  place  is  a  katabolic  wasting  away, 
which  soon  proves  destructive,  and  the  higher  the 
temperature  the  more  rapidly  the  fatal  result  is  reached. 
A  frog  in  winter  lives  at  the  bottom  of  a  pond  breath- 
ing only  through  its  skin  and  eating  not  at  all,  but  as 
soon  as  the  temperature  rises  it  must  eat  and  breathe 
through  its  lungs  or  perish.  It  is  quite  true  that  even 
in  ice  40  per  cent  of  typhoid  bacilli  perish  in  3  hours 
and  98  per  cent  in  2  weeks  (Sedgwick  and  Winslow, 
1902).  Recent  work  has  shown,  however,  that  they 
die  in  spite  of  the  cold,  not  on  account  of  it,  and  that 
the  decrease  is  more  rapid  at  higher  temperatures, 
unless  of  course  food-supply  and  other  conditions  admit 
of  multiplication.  Houston  (1911)  has  furnished  a 
very  clear  demonstration  of  this  temperature  rela- 
tion by  storing  typhoid  bacilli  in  water  with  the  results 
tabulated  on  page  22. 


22 


ELEMENTS  OF  WATER  BACTERIOLOGY 


EFFECT  OF  TEMPERATURE  ON  SURVIVAL  OF  TYPHOID 
BACTERIA  IN  WATER 

(HOUSTON,  1911) 


Temperature  C. 

Percentage  of  Typhoid 
Bacilli  Surviving   after 
One  Week. 

Period  of  Final 
Disappearance  of  BacilH. 

O  

46 

9  weeks 

c; 

14 

7  weeks 

jo 

O  O7 

5  weeks 

18  

O.O4 

4  weeks 

Ruediger  (1911)  has  shown  that  colon  bacilli  are 
far  more  abundant  in  the  Red  Lake  River  during  the 
winter  when  the  river  is  covered  with  ice  than  in  sum- 
mer, although  the  volume  of  the  river  and  the  amount 
of  sewage  pollution  are  about  the  same.  Typhoid 
bacilli  in  celloidin  dialyzers  floated  down  the  river 
showed  only  2.5  and  3.5  per  cent  surviving  in  2  days 
and  0.51,  0.89,  2.2  and  3.2  per  cent  surviving  in  3  days 
when  the  river  was  not  frozen,  while  dialyzers  suspended 
through  the  ice  in  colder  weather  showed  6.1,  10.5,  17.7, 
46.8  and  62.9  per  cent  surviving  in  five  different  experi- 
ments after  2  days,  31  per  cent  in  3  days,  19  per  cent 
in  7  days,  and  2.5  per  cent  in  14  days.  Ruediger 
attributes  this  greater  persistence  at  low  temperatures 
to  the  absence  of  poisonous  waste  products  of  other 
organisms  and  to  protection  from  the  light;  but  there 
can  be  little  doubt  that  it  is  mainly  a  result  of  the  general 
preservative  effect  of  cold.  From  an  epidemiological 
standpoint  the  conclusion  that  disease  germs  perish 
quickly  in  warm  waters  is  amply  confirmed.  Almost 
without  exception  outbreaks  of  typhoid  fever  due  to 


THE  BACTERIA  IN  NATURAL  WATERS          23 

polluted  water  occur  in  cold  weather  and  this  is,  in 
part  at  least,  due  to  the  greater  persistence  of  typhoid 
bacilli  at  low  temperatures. 

Relation  between  Time  of  Storage  and  Self-purifica- 
tion. It  is  obvious  that  the  efficiency  of  all  the  agencies 
which  tend  to  decrease  the  number  of  bacteria  in  sur- 
face waters  will  increase  with  the  prolongation  of  the 
period  for  which  they  act.  Time  is  the  great  measure 
of  self -purification. 

The  longer  the  storage  the  greater  the  improvement, 
and  after  a  certain  period  even  a  fairly  polluted  water 
may  be  safe  and  potable.  The  absolute  time  necessary 
to  produce  this  result  varies  of  course  according  to  many 
conditions.  Food  supply,  light,  temperature  and  the 
activity  of  other  living  forms  vary  widely  and  in  depos- 
ited material  conditions  are  different  from  those  which 
obtain  in  the  water  itself.  Jordan,  Russell  and  Zeit 
(1904),  in  an  important  series  of  experiments,  added 
typhoid  bacilli  to  the  unsterilized  waters  of  Lake 
Michigan,  the  Chicago  River  and  Drainage  Canal  and 
the  Illinois  River,  in  collodion  sacs  suspended  in  the 
respective  bodies  of  water.  From  the  relatively  pure 
Lake  Michigan  water  the  specific  organisms  could  be 
isolated  for  at  least  a  week,  but  in  the  polluted  waters 
of  the  rivers  and  the  Drainage  Canal  they  were  not 
found  after  3  days  except  in  a  single  instance.  Russell 
and  Fuller,  (1906)  confirmed  these  general  results, 
finding  that  typhoid  bacilli  would  live  for  10  days  in 
the  unsterilized  water  of  Lake  Mendota,  while  they 
could  be  isolated  only  after  5  days  when  immersed 
in  sewage.  Other  observers  record  much  greater 


24         ELEMENTS  OF  WATER  BACTERIOLOGY 

viability  for  the  typhoid  bacillus.  Savage  (1905) 
added  a  heavy  dose  of  the  organism  to  unsterilized 
tidal  mud  and  found  it  living  after  5  weeks.  Hoffmann 
(1905),  after  inoculating  a  large  aquarium  with  a  rich 
typhoid  culture,  was  able  to  isolate  the  germ  from  the 
water  after  four  weeks  and  from  the  mud  at  the  bottom 
after  two  months.  Konradi  (1904)  reports  the  per- 
sistence of  typhoid  bacilli  in  unsterilized  tap  water 
for  over  a  year. 

These  last  experiments  deal  only  with  the  maximum 
survival  period  for  a  few  out  of  great  numbers  of  germs 
introduced  into  the  water  or  mud,  and  entirely  ignore 
the  quantitative  aspects  of  the  case.  When  one  con- 
siders the  proportion  of  the  original  bacteria  surviving, 
the  period  necessary  to  bring  about  a  reasonably  safe 
condition  is  found  to  be  much  shorter.  Houston 
(1908)  has  shown  that  when  water  is  artificially  infected 
with  .typhoid  bacilli  and  stored,  99.9  per  cent  of  the 
disease  germs  perish  in  one  week,  although  some  may 
persist  for  from  i  to  9  weeks. 

In  later  experiments  (Houston,  1911)  he  finds  that 
"  uncultivated "  typhoid  bacilli  added  to  the  water 
directly  from  the  urinary  sediment  of  a  disease  carrier 
perish  much  more  rapidly  than  the  laboratory  strains, 
usually  disappearing  entirely  after  one  week  and  always 
after  three.  On  a  number  of  occasions  Dr.  Houston 
gave  dramatic  expression  to  his  confidence  in  these 
negative  laboratory  findings  by  drinking  half  pint 
portions  of  water  which  a  few  weeks  previously  had 
contained  millions  of  typhoid  bacilli.  We  have  plenty 
of  practical  epidemiological  evidence,  such  as  that 


THE  BACTERIA  IN  NATURAL  WATERS          25 

offered  in  the  Chicago  Drainage  Canal  case  and  in  the 
lawsuit  over  the  condition  of  the  water  supply  of  Jersey 
City,  to  confirm  the  general  conclusion  that  any  water 
which  has  been  stored  for  4  weeks  is  practically  safe. 

Bacteria  in  Ground-waters.  In  general  we  have 
seen  that  surface-waters  tend  continually  to  decrease 
in  bacterial  content  after  their  first  period  of  contact 
with  the  humus  layer  of  the  soil.  In  that  other  portion 
of  the  meteoric  water  which  penetrates  below  the 
surface  of  the  earth  to  join  the  reservoir  of  ground- 
water,  later  to  reappear  as  the  flow  of  springs  and  wells, 
this  diminution  is  still  more  marked,  since  the  filtering 
action  of  the  earth  removes  not  only  most  of  the  bac- 
teria, but  much  of  their  food  material  as  well.  The 
numbers  of  bacteria  in  the  soil  itself  decrease  rapidly 
as  one  passes  downward.  Kabrhel  (1906)  found  several 
million  per  c.c.  in  surface  samples  of  woodland  soil, 
a  few  thousands  or  tens  of  thousands  half  a  meter 
below,  and  usually  only  hundreds  in  centimeter 
samples  collected  at  depths  greater  than  a  meter. 
Many  observers  formerly  believed  that  all  ground- 
waters  were  nearly  free  from  bacteria,  because  often 
no  colonies  appeared  on  plates  counted  after  the 
ordinary  short  periods  of  time.  If,  however,  a  longer 
period  of  incubation  be  adopted  considerable  numbers 
may  be  obtained. 

For  convenience  we  may  divide  ground-waters  into 
three  groups,  namely:  shallow  open  wells,  springs  and 
"  tubular "  (driven)  or  deep  wells.  This  division  is 
important  because  ordinary  shallow  wells  form  a  group 
by  themselves  in  respect  to  the  possibility  of  aerial  and 


26         ELEMENTS  OF  WATER  BACTERIOLOGY 

surface  contamination,  their  water  often  being  fairly 
rich  in  bacterial  life.  Egger  (Wolff  hugel,  1886)  examined 
60  wells  in  Mainz  and  found  that  17  of  them  contained 
over  200  bacteria  to  the  cubic  centimeter.  Maschek 
(Maschek,  1887)  found  36  wells  out  of  48  examined  in 
Leitmeritz  which  had  a  bacterial  content  of  over  500 
per  c,c.  Fischer  (Horrocks,  1901)  reported  120  wells 
in  Kiel  which  gave  over  500  bacteria  per  c.c.  and  only 
51  with  less  than  that  number. 

In  the  examination  of  147  shallow  farmyard  wells 
by  one  of  us  (S.  C.  P.)  it  was  found  that  124  of  the 
wells  which  contained  no  B.  coli,  and  were  therefore 
probably  free  from  fecal  pollution,  averaged  190  bacilli 
per  c.c.  while  23  which  gave  positive  tests  for  B.  coli 
averaged  570  per  c.c.  The  distribution  of  the  two  series 
of  samples  according  to  the  number  of  bacteria  present 
is  indicated  in  the  table  below. 


BACTERIA  IN  SHALLOW  FARMYARD  WELLS 
PERCENTAGE  OF  SAMPLES  IN  EACH  GROUP 


Bacteria  per  c  c  

0 

i- 

ii- 

21- 

5i- 

101- 

501- 

IOOI- 

2OOI- 

10 

20 

50 

IOO 

500 

IOOO 

2OOO 

3000 

Series  I.  B.  coli  absent. 

3 

16 

U 

16 

II 

31 

5 

4 

Series  II.  B.  coli  present  . 

5 

IO 

57 

IO 

14 

5 

Very  similar  results  are  reported  for  shallow  wells 
used  as  farm  water-supplies  in  Minnesota  by  Kellerman 
and  Whittaker  (1909),  although  the  general  quality 
of  the  wells  examined  was  considerably  below  that 
of  the  series  tabulated  above. 


THE  BACTERIA  IN  NATURAL  WATERS 


27 


In  the  ordinary  standard  48-hour  period  very  few 
bacteria  develop  from  normal  spring- waters.  Thus 
in  an  examination  of  spring-waters  made  by  the  Mas- 
sachusetts State  Board  of  Health  in  1900  (Massachusetts 
State  Board  of  Health,  1901),  of  37  springs  which  were 
practically  unpolluted  and  had  less  than  o.io  part 
per  100,000  excess  of  chlorine  over  the  normal,  54  sam- 
ples were  examined  and  gave  an  average  of  41  bacteria 
per  c.c.  Only  6  samples  showed  figures  over  50. 

It  now  remains  to  consider  the  other  great  division 
of  ground-waters,  namely,  deep,  "  driven,"  or  "  tubular  " 
wells,  which,  if  carefully  constructed,  should  ordinarily 
be  free  from  all  surface-water  contamination,  and  should 
show  low  bacterial  counts.  The  results  tabulated  below 
obtained  by  Houston  in  the  examination  of  a  series  of 
deep  wells  of  high  quality  at  Tunbridge  Wells  are 
fairly  typical. 

BACTERIAL  CONTENT  OF  DEEP  WELL  WATERS 

(HOUSTON,  1903) 
Bacteria  per  c.c. 


36 

6 

9 

4 

i 

16 

17 

4 

3 

12 

2 

4 

10 

5 

2 

Fifteen  driven  wells  in  the  neighborhood  of  Boston, 
examined  in  1903,  showed  at  the  end  of  48  hours  an 
average  of  only  18  colonies  per  c.c.;  and  the  results 
of  certain  examinations  of  other  wells  and  springs, 
recently  made  by  the  authors,  are  given  in  the  table 
on  page  28. 


28         ELEMENTS  OF  WATER  BACTERIOLOGY 
BACTERIA  IN  DEEP  WELL  AND  SPRING  WATERS 


Town 

Bacteria 
per  c.c. 

Town. 

Bacteria 
per  c.c. 

Worcester,  Mass  
Waltham,  Mass  
Newport,  R.I  

IO 

3 

7 

Saranac  Lake,  N.  Y.   . 
Ellenville,  N.  Y  
Hyde  Park,  Mass  

II 

o 

12 

It  is  plain  that  water  absolutely  free  from  bacteria 
is  not  ordinarily  obtained  from  any  source.  In  deep 
wells,  however,  their  number  is  small;  and  the  peculiar 
character  of  the  organisms  present  is  manifested  in 
many  cases  by  the  slow  development  at  room  tem- 
perature (frequently  no  growth  until  the  third  day), 
the  entire  absence  of  liquefying  colonies,  and  the 
abundance  of  chromogenic  species. 


CHAPTER  II 

THE  QUANTITATIVE  BACTERIOLOGICAL  EXAMINATION 
OF  WATER 

Relation  of  the  Medium  to  the  Number  of  Bacteria 
Obtained.  The  customary  methods  for  determining 
the  number  of  bacteria  in  water  do  not  reveal  the  total 
bacterial  content,  but  only  a  very  small  fraction  of  it, 
as  becomes  apparent  when  we  consider  the  large  num- 
ber of  organisms,  nitrifying  bacteria,  strict  anaerobes, 
etc.,  which  refuse  to  grow,  or  grow  only  very  slowly  in 
ordinary  culture  media,  and  which,  therefore,  escape 
detection.  On  the  one  hand,  certain  obligate  parasites 
cannot  thrive  in  the  absence  of  the  rich  fluids  of  the 
animal  body;  on  the  other  hand,  the  prototrophic 
bacteria,  adapted  to  the  task  of  wrenching  energy  from 
nitrites  and  ammonium  compounds  are  unable  to  develop 
in  the  presence  of  so  much  organic  matter.  Winslow 
(1905)  in  the  examination  of  sewage  and  sewage  effluents, 
found  20-70  times  as  many  bacteria  by  microscopic 
enumeration  as  by  the  gelatin  plate  count.  Certain 
special  media  enable  us  to  obtain  much  larger  counts 
than  those  yielded  by  the  ordinary  gelatin  method. 
The  Nahrstoff  Heyden  agar,  for  example,  has  been 
strongly  advocated  by  Hesse  (Hesse  and  Niedner, 
1898)  and  other  German  bacteriologists  upon  this 

29 


30         ELEMENTS  OF  WATER  BACTERIOLOGY 

ground.  In  this  country  Gage  and  Phelps  (Gage  and 
Phelps,  1902)  showed  that  the  numbers  obtained  by 
the  ordinary  procedure  were  only  from  5  to  50  per  cent 
of  those  obtained  by  the  use  of  Heyden's  Nahrstoff 
agar.  For  practical  sanitary  purposes,  however,  our 
methods  are  fairly  satisfactory.  Within  limits,  it  is 
of  no  great  importance  that  one  method  allows  the 
growth  of  more  bacteria  than  another.  When  we  are 
using  the  quantitative  analysis  as  a  measure  of  sewage 
pollution  the  essential  thing  is  that  the  section  of  the 
total  bacterial  flora  which  we  obtain  should  be  thor- 
oughly representative  of  that  portion  of  it  in  which 
we  are  most  interested- — the  group  of  the  quickly 
growing,  rich-food-loving  sewage  forms.  In  this  respect 
meat-gelatin-peptone  appears  to  be  unrivalled;  and  it 
is  in  this  respect  that  such  media  as  Nahrstoff  agar  fail. 
Miiller  (1900)  showed  that  the  larger  counts  obtained 
by  plating  on  the  Nahrstoff  medium  are  due  to  the 
fact  that  it  specially  favors  the  more  prototrophic 
forms,  among  the  water  bacteria  themselves.  Intestinal 
organisms  and  even  the  ordinary  putrefactive  germs, 
when  plated  in  pure  culture,  show  no  higher  counts  on 
Nahrstoff  agar  than  on  gelatin.  Gage  and  Adams 
(1904)  found  by  plating  pure  cultures  of  the  common 
laboratory  bacteria,  saprophytes  and  parasites,  that 
Nahrstoff  counts  were  actually  lower  than  those  obtained 
by  the  use  of  gelatin.  When  sewage  and  highly  polluted 
waters  are  examined  counts  are  slightly  higher  on 
Nahrstoff  media,  while  with  purer  waters  the  Nahrstoff 
numbers  are  far  in  excess  of  those  obtained  with  gelatin. 
Winslow  (1905)  found  the  ratio  of  Nahrstoff  agar 


QUANTITATIVE  EXAMINATION  OF  WATER     31 


to  gelatin  count  to  be  1.7  to  i.o  for  sewage,  and  4.8 
to  i.o  for  sand  filter  effluent.  With  waters  of  still 
better  quality  the  ratio  goes  up  higher,  reaching  a 
maximum  when  the  bacteria  which  increase  and 
multiply  in  water  are  most  abundant.  Miiller  (1900) 
found,  for  example,  that  water  which  normally  showed 
six  times  as  many  bacteria  on  Nahrstoff  agar  as  on 
gelatin  might  give  a  Nahrstoff-gelatin  ratio  of  20-30 
after  it  had  been  standing  for  some  time  in  the  supply 
pipes.  The  table  below,  taken  from  the  valuable 
paper  by  Gage  and  Phelps  (1902),  shows  strikingly 
the  different  Nahrstoff-agar  ratios  for  waters  of 

TABLE  SHOWING  PERCENTAGES  OF  BACTERIA  DEVELOP- 
ING ON  REGULAR  AGAR  AND  NAHRSTOFF  AGAR 
FOR  DIFFERENT  CLASSES  OF  WATERS 

(GAGE  AND  PHELPS,  1902) 

Regular  Agar 


Days'  Count. 

Class  of  Water. 

2 

3 

4 

5 

6 

7 

8 

Ground  water  

o 

5 

6 

6 

6 

6 

6 

Filtered  water  

6 

7 

7 

7 

7 

7 

7 

Merrimac  River.  .  . 

6 

7 

7 

8 

8 

9 

9 

Filtered  sewage.  .  .  . 

14 

17 

18 

iQ 

iQ 

19 

19 

Sewage  

34 

44 

46 

46 

46 

46 

46 

Narhstoff  Agar 


Ground  water  

6 

43 

78 

88 

93 

IOO 

IOO 

Filtered  water  

37 

69 

80 

92 

98 

IOO 

IOO 

Merrimac  River  .  .  . 

29 

78 

93 

97 

97 

99 

IOO 

Filtered  sewage.  .  .  . 

26 

65 

93 

95 

97 

99 

IOO 

Sewage  

39 

75 

oc 

IOO 

IOO 

IOO 

IOO 

32         ELEMENTS  OF  WATER  BACTERIOLOGY 

various  grades  of  purity.  It  is  obvious  from  all  these 
facts  that  the  effect  of  using  the  Nahrstoff  medium 
is  to  increase  disproportionately  the  bacterial  counts 
obtained  from  purer  waters  and  thus  to  diminish  the 
difference  in  bacterial  content  between  normal  and 
contaminated  sources.  The  ordinary  agar  and  gelatin 
media,  on  the  other  hand,  are  adapted  to  the  growth 
of  intestinal  and  putrefactive  forms  and,  therefore, 
serve  best  the  prime  object  of  bacteriological  water 
examination. 

The  first  requisite  in  a  procedure  for  water  analysis 
is,  then,  that  it  should  be  adapted  to  the  end  in  view, 
the  differentiation  of  pure  and  contaminated  waters. 
The  second  and  equally  important  requirement  is  that 
the  procedure  should  be  a  standard  one,  so  that  results 
obtained  at  different  times  and  by  different  observers 
may  be  comparable.  In  this  respect  the  work  of  G.  W. 
Fuller,  G.  C.  Whipple,  and  other  members  of  the 
Committee  on  Standard  Methods  of  the  American  Public 
Health  Association  has  placed  the  art  of  quantitative 
water  analysis  in  this  country  in  a  very  satisfactory 
state  by  contrast  with  the  varying  practices  which 
prevail  in  England  and  Germany.  The  first  report  on 
this  question  was  made  in  1897  (Committee  of  Bac- 
teriologists, 1898).  A  permanent  Committee  on  Stand- 
ard Methods  was  then  formed  which  reported  in  1901 
(Fuller,  1902),  in  1904  (Committee  on  Standard  Methods 
of  Water  Analysis,  1905),  and  again  in  1911  (Committee 
on  Standard  Methods  for  the  Examination  of  Water 
and  Sewage,  1912),  recommending  in  considerable 
detail  a  standard  routine  procedure  for  the  quantitative 


QUANTITATIVE  EXAMINATION  OF  WATER     33 

and  qualitative  bacteriological  examination  of  water 
for  sanitary  purposes.  These  reports  have  had  a  far- 
reaching  effect  in  simplifying  and  unifying  the  methods 
of  water  analysis.  Similar  results  have  followed  from 
the  work  of  the  English  Committee  appointed  to  con- 
sider the  Standardization  of  Methods  for  the  Bac- 
terioscopic  Examination  of  Water  which  reported  in 
1904,  although  this  committee  unfortunately  did  not 
consider  the  process  of  media  making  in  great  detail. 
The  last  report  of  the  American  Committee  on  Standard 
Methods  (1912)  will  be  adhered  to  in  this  and  succeed- 
ing chapters  unless  otherwise  specifically  stated;  and 
that  portion  of  its  report  which  deals  with  methods  of 
making  media  will  be  found  in  full  in  the  appendix. 

Standard  Procedure  for  Quantitative  Determination  of 
Bacteria  in  Water.  The  procedure  for  the  quantitative 
determination  of  bacteria  in  water  consists,  in  brief, 
in  mixing  a  definite  amount  of  a  suitably  collected 
specimen  of  the  water  with  a  sterile,  solidifiable  culture 
medium  and  incubating  it  for  a  sufficiently  long  time 
to  permit  reproduction  of  the  bacteria  and  the  forma- 
tion of  visible  colonies  which  may  be  counted.  The 
process  is  divided  naturally  into  four  stages — sampling, 
plating,  incubating,  and  counting. 

Sampling.  All  samples  of  water  for  bacteriological 
examination  should  be  collected  in  clean,  sterile  bottles 
with  wide  mouths  and  glass  stoppers,  preferably  of  the 
flat  mushroom  type.  It  is  desirable  that  these  bottles 
should  have  a  capacity  of  at  least  100  c.c. 

They  should  be  cleaned  thoroughly  before  using,  by 
treatment  with  sulphuric  acid  and  potassium  bichromate 


34         ELEMENTS  OF  WATEE  BACTERIOLOGY 

or  with  alkaline  permanganate  of  potash  followed  by 
sulphuric  acid,  dried  by  draining,  and  sterilized  by 
dry  heat  at  160°  C.  for  at  least  i  hour,  or  by  steam  at 
115-120°  for  15  minutes.  If  not  to  be  used  immediately 
the  neck  and  stopper  should  be  protected  against  dust 
or  other  contamination  by  wrapping  with  lead-foil. 
For  transportation  the  bottle  should  be  enclosed  in  a 
suitable  case  or  box. 

The  greatest  care  must  be  taken  that  the  fingers  do 
not  touch  the  inside  of  the  neck  of  the  bottle  or  the  cone 
of  the  stopper,  as  the  water  thereby  would  become 
seriously  contaminated  and  rendered  unfit  for  examina- 
tion. It  is  well  known  that  bacteria  are  found  abun- 
dantly upon  the  skin,  and  Winslow  (Winslow,  1903) 
has  shown  that  even  B.  coli  is  present  upon  the  hands 
in  a  considerable  number  of  cases. 

In  order  to  obtain  a  fair  sample,  great  precautions 
must  be  taken,  and  these  will  vary  with  the  different 
classes  of  waters  to  be  examined  and  with  local  condi- 
tions. If  a  sample  is  to  be  taken  from  a  tap,  the  water 
should  be  allowed  to  flow  at  least  five  minutes  (if  from 
a  tap  in  regular  use)  or  for  a  longer  period  in  case  the 
water  has  been  standing  in  the  house-service  system. 
In  the  small  pipes,  changes  in  bacterial  content  are 
liable  to  occur,  certain  species  dying  and  others  mul- 
tiplying. 

If  a  sample  is  to  be  taken  from  a  pump  similar  pre- 
cautions are  necessary.  The  pump  should  be  in  con- 
tinuous operation  for  5  minutes  at  least,  and  preferably 
for  half  an  hour  before  the  sample  is  taken,  in  order  to 
avoid  excessively  high  numbers  due  to  the  growth  of 


QUANTITATIVE  EXAMINATION  OF  WATER      35 

bacteria  within  the  well  and  pump,  the  bacterial  con- 
dition of  the  water  as  it  passes  through  the  ground  being 
what  we  wish  to  determine.  Thus  Heraeus  (Heraeus, 
1886)  in  a  well-water  which  had  been  but  little  used 
during  the  preceding  36  hours  found  5000  organisms 
per  c.c.;  when  the  well  was  emptied  by  continuous 
pumping,  a  second  sample,  after  an  interval  of  half  an 
hour,  gave  only  35.  Maschek  (Tiemann  and  Gartner, 
1889)  obtained  similar  results,  shown  in  the  following 
table: 

EFFECT  OF  PUMPING  ON  THE  BACTERIAL  CONTENT  OF 
WELL-WATER 

Well-water  after  continuous  pumping  for  fifteen  minutes    .  .  458 

many  hours 140 

later 68 

after  continuous  pumping  for  fifteen  minutes    .  .  578 

many  hours 1 79 

later 73 

After  a  proper  interval  of  pumping  the  sample  of  a 
well-water  may  be  collected  from  the  pet-cock  of  the 
pump  or  from  a  near-by  tap.  With  a  hand-pump, 
such  as  is  found  in  domestic  shallow  wells,  the  water  is, 
of  course,  pumped  directly  into  the  sample  bottle. 
The  difficulties  in  securing  an  average  sample  from 
this  latter  source  are  often  great,  since  if  the  flooring 
about  the  pump  is  not  tight,  as  is  usually  the  case,  con- 
tinued pumping  may  wash  in  an  unusual  amount  of 
surface  pollution. 

In  sampling  surface-waters,  the  greatest  precautions 
must  be  observed  to  prevent  contamination  from  the 
fingers.  In  still  waters  the  fairest  sample  is  one  taken 


36         ELEMENTS  OF  WATER  BACTERIOLOGY 

from  several  inches  down,  as  the  surface  itself  is  likely 
to  have  dust  particles  floating  upon  it.  The  method 
most  frequently  recommended  is  to  plunge  the  bottle 
mouth  downward  to  a  depth  of  a  foot  or  so,  then 
invert  and  allow  the  bottle  to  fill. 

Whenever  any  current  exists,  the  mouth  of  the  bottle 
should  be  directed  against  it  in  order  to  carry  away  any 
bacteria  from  the  fingers.  If  there  is  no  current,  a 
similar  effect  can  be  produced  by  turning  the  bottle 
under  water  and  giving  it  a  quick  forward  motion.  In 
rapidly  flowing  streams  it  is  only  necessary  to  hold 
the  bottle  at  the  surface  with  the  mouth  pointed 
up-stream. 

For  taking  samples  of  water  at  greater  depths,  a 
number  of  devices  have  been  employed,  all  of  which 
are  fairly  satisfactory.  The  essentials  are,  first,  a  weight 
to  carry  the  bottle  down  to  the  desired  depth,  and, 
second,  some  method  of  removing  the  stopper  when 
that  depth  is  reached.  The  student  will  find  one  good 
form  of  apparatus  described  in  Abbott's  "  Principles 
of  Bacteriology"  (Abbott,  1899);  an  admirable  one 
was  devised  by  Hill  and  Ellms  (Hill  and  Ellms,  1898); 
and  Thresh  (1904)  figures  an  ingenious  device  for  the 
same  purpose.  Miquel  and  Cambier  (Miquel  and 
Cambier,  1902)  and  other  authors  recommend  the  use 
of  a  sealed  glass  bulb  with  a  capillary  tube  which  can 
be  broken  off  at  the  desired  moment.  Drew  (1912) 
has  devised  an  interesting  sampling  apparatus  for  use 
at  great  depths  in  the  sea. 

Changes   in  Bacterial  Numbers  after  Sampling.    As 
soon  as  a  sample  of  water  is  collected  its  conditions 


QUANTITATIVE  EXAMINATION  OF  WATER      37 

of  equilibrium  are  upset  and  a  change  in  the  bacterial 
content  begins.  Even  in  the  purest  spring-waters, 
which  contain  but  few  bacteria  when  collected,  and  in 
which  the  amount  of  organic  matter  is  infinitesimal, 
enormous  numbers  may  be  found  after  storage  under 
laboratory  conditions  for  a  few  days  or  even  a  few  hours. 
In  some  cases  the  rise  in  numbers  is  gradual,  in  others 
very  rapid.  The  Franklands  (Frankland,  1894)  record 
the  case  of  a  deep-well  water  in  which  the  bacteria 
increased  from  7  to  495,000  in  3  days.  Miquel  (Miquel, 
1891)  from  his  researches,  arrived  at  the  conclusion 
that  in  surface-waters  the  rise  is  less  rapid  than  in  waters 
from  deep  wells  or  springs,  and  that  in  the  latter  case 
the  decrease,  after  reaching  a  maximum,  is  likewise 
rapid  and  steady.  Just  how  far  protection  from  light, 
increase  in  temperature,  and  a  destruction  of  higher 
micro-organisms  is  responsible  for  the  increase,  and 
to  what  extent  an  exhaustion  of  food-supply  or  the 
formation  of  toxic  waste  products  causes  the  succeeding 
decrease,  we  are  not  aware;  but  the  facts  are  well 
established. 

Whipple  has  exhaustively  studied  the  details  of 
this  multiplication  of  bacteria  in  stored  waters 
and  has  shown  in  the  table  given  below  that 
there  is  first  a  slight  reduction  in  the  number 
present,  lasting  perhaps  for  6  hours;  followed  by 
the  great  increase  noted  by  earlier  observers.  It 
is  probable  that  there  is  a  constant  increase  of  the 
typical  water  bacilli,  overbalanced  at  first  by  a 
reduction  in  other  forms,  for  which  the  environment 
is  unsuitable. 


38 


ELEMENTS  OF  WATER  BACTERIOLOGY 


BACTERIAL   CHANGES   IN  WATER   DURING  STORAGE 
(WHIPPLE,  1901) 


Sample 

Initial 
Temper- 
ature. 

Temp, 
of  Incu- 
bation of 
Sample. 

Number  of  Bacteria  per  c.c. 

Initial. 

After 
3  Hours. 

After 
6  Hours. 

After 
24  Hours. 

After 
48  Hours. 

C. 

C. 

A 

7.6° 

17.0° 

260 

215 

230 

900 

27,000 

B 

7-6° 

17.0° 

260 

245 

255 

720 

10,850 

C 

7.6° 

12.5° 

260 

270 

231 

600 

2,790 

D 

7.6° 

12.5° 

260 

270 

245 

710 

1,  800 

E 

7.6° 

2.4° 

260 

243 

210 

675 

1,980 

F 

7.6° 

2-4° 

260 

235 

270 

560 

1,980 

G 

11.0° 

12.8° 

77 

55 

58 

101 

10,250 

H 

11.0° 

12.8° 

77 

53 

74 

87 

2,175 

I 

11.0° 

23.6° 

77 

5i 

52 

11,000 

41,400 

J 

6-7° 

20.0° 

430 

375 

245 

385,ooo1 

K 

6.7° 

20.0° 

430 

345 

405 

75o,ooo1 

L 

23-2° 

23.0° 

510 

340 

230 

8,000 

20,000 

M 

23-2° 

2-5° 

525 

300 

220 

380 

2,200 

1  0.0005  Per  cent  peptone  added  to  the 


water. 


WolfThiigel  and  Riedel  (Wolffhiigel  and  Riedel, 
1886)  noted  the  dependence  of  this  multiplication 
on  the  air-supply,  vessels  closed  with  rubber  stoppers 
showing  lower  numbers  than  those  plugged  with  cotton. 
Similarly,  Whipple  found  that  the  multiplication  of 
bacteria  was  much  greater  when  bottles  were  only 
half  full  than  when  they  were  filled  completely;  and  also, 
as  shown  in  the  very  striking  table  on  page  39,  that 
the  size  of  the  bottle  markedly  influenced  the  growth. 

An  important  series  of  investigations  by  Kohn  (1906) 
suggests  that  this  phenomenon  of  multiplication  dur- 
ing storage  may  be  due  in  part  to  the  solution  of  certain 
constituents  of  glass  which  favor  bacterial  life,  since  the 
increase  is  notably  greater  in  bottles  of  the  more  soluble 
glasses. 


QUANTITATIVE  EXAMINATION  OF  WATER      39 


EFFECT  OF  SIZE  OF  VESSEL  UPON  THE  MULTIPLICATION 
OF  WATER  BACTERIA  DURING  STORAGE 

(WHIPPLE,  1901) 


Sample 

Bottle. 

Temp, 
of 
Incuba- 
tion. 

Number  of  Bacteria  per  c.c. 

Ini- 
tial.1 

After 
3  Hrs. 

After 
6  Hrs. 

After 
12  Hrs 

After 
24  Hrs. 

After 
48  Hrs. 

C 

A 

i  -gallon 

13° 

77 

63 

65 

47 

42 

175 

B 

2-qutirt 

13° 

77 

59 

63 

60 

45 

690 

C 

i-quart 

13° 

77 

63 

63 

47 

46 

325 

D 

i-pint 

13° 

77 

57 

61 

36 

38 

630 

E 

2-ounce 

13° 

77 

55 

58 

47 

IOI 

10.250 

F 

i  -gallon 

24° 

77 

81 

97 

275 

290 

300 

G 

2-quart 

24° 

77 

92 

59 

62 

1  80 

250 

H 

i-quart 

24° 

77 

84 

77 

46 

340 

900 

I 

i-pint 

24° 

77 

51 

46 

100 

2,950 

7,O2O 

J 

2-ounce 

24° 

77 

5i 

52 

U5 

11,000 

41,400 

1  Average  of  five  plates. 

Whipple's  table,  quoted  above,  shows  that  the  multi- 
plication during  storage  was  greater  at  a  higher  tem- 
perature; and  this  is  a  well-recognized  general  rule. 
In  order  to  obviate  the  abnormal  results  of  storage 
increase  it  is  therefore  obvious  that  samples  must  be 
examined  shortly  after  collection  and  that  they  must 
be  kept  cool  during  their  necessary  storage.  If  fairly 
pure  waters  are  placed  upon  ice  and  kept  between  o 
degrees  and  10  degrees,  they  will  show  no  material 
increase  in  12  hours.  With  polluted  water,  however, 
another  danger  is  here  introduced.  Samples  of  such 
water  when  packed  in  ice  show  a  marked  decrease 
due  to  the  large  number  of  sensitive  intestinal  bacteria 
present.  Jordan  (Jordan,  1900)  found  that  three 
samples  of  river-water  packed  in  ice  for  48  hours  fell 


40         ELEMENTS  OF  WATER  BACTERIOLOGY 

off  from  535,000  to  54,500;  from  412,000  to  50,500, 
and  from  329,000  to  73,000,  respectively.  It  is,  there- 
fore, important  that  even  iced  samples  should  not  be 
kept  too  long;  and  it  is  desirable  to  adhere  strictly 
to  the  recommendations  of  the  Standard  Methods 
Committee  that  the  interval  between  sampling  and 
examination  should  not  exceed  12  hours  in  the  case  of 
relatively  pure  waters,  6  hours  in  the  case  of  relatively 
impure  waters,  and  i  hour  in  the  case  of  sewage. 

Plating.  The  bottle  containing  the  sample  of  water 
is  first  shaken  at  least  twenty-five  times  in  order  to 
get  an  equal  distribution  of  the  bacteria.  If  the  num- 
ber of  bacteria  present  is  probably  not  greater  than  200, 
i  c.c.  is  then  withdrawn  with  a  sterile  i  c.c.  pipette 
and  delivered  into  a  sterile  Petri  dish  of  10  cm.  diameter. 
To  this  is  added  5  c.c.  of  standard  10  per  cent  gelatin 
at  a  temperature  of  about  30°  C.,  or  standard  agar 
(7  c.c.)  at  40-42°  C.  Should  the  number  of  bacteria 
per  c.c.  probably  exceed  200,  dilution  is  necessary. 
This  is  best  accomplished  by  adding  i  c.c.  of  the  water 
in  question  to  9,  99  or  999,  etc.,  c.c.  of  sterile  tap  water 
according  to  the  amount  of  dilution  required.  The 
diluted  sample  is  then  shaken  thoroughly  and  i  c.c. 
taken  for  enumeration.  In  order  to  determine  the 
number  of  bacteria  originally  present  it  is  only  neces- 
sary to  multiply  by  the  factor  10,  100,  or  1000,  etc. 

When  a  sample  of  water  from  an  unknown  source 
is  to  be  examined  it  is  generally  desirable  to  make 
two  check  plates  at  each  of  the  above  dilutions,  select- 
ing those  which  give  nearest  to  200  colonies  on  the 
plates  after  incubation  as  the  ones  on  which  to  rely 


QUANTITATIVE  EXAMINATION  OF  WATER      41 

for  the  count.  A  much  smaller  number  will  not  give 
average  figures,  and  if  more  than  200  colonies  are  present 
on  a  plate  many  bacteria  will  be  checked  by  the  waste 
products  of  those  which  first  develop  and  the  count 
obtained  will  be  too  low.  After  the  addition  of  the 
diluted  sample  and  the  nutrient  medium,  their  thorough 
mixture  in  an  even  layer  on  the  bottom  of  the  plate 
is  obtained  by  careful  tipping  and  rotation. 

It  was  formerly  customary  to  mix  the  water  with  the 
gelatin  in  the  tube  before  pouring  into  the  plate,  but 
this  method  is  objectionable  because  there  is  always 
a  residuum  of  medium  remaining  in  the  tube  which 
will  retain  varying  numbers  of  bacteria  and  thus 
interfere  with  the  accuracy  of  the  count.  Before  pour- 
ing the  medium  into  the  plate  the  mouth  of  the  tube 
should  be  flamed  to  remove  any  possibility  of  con- 
tamination. 

The  usual  method  of  determining  the  number  of 
bacteria  in  water  for  sanitary  purposes  in  Germany, 
England  and  the  United  States  has  always  been  by 
the  use  of  gelatin  plates  with  a  2 -day  incubation  period 
at  20  degrees.  The  1905  Standard  Methods  Report 
of  the  American  Public  Health  Association  Committee 
recommended  this  procedure,  which  has  been  universally 
adopted.  The  1912  Report,  however,  suggests  the  use 
of  agar  with  a  i-day  period  at  37  degrees,  as  yielding 
quicker  results  and  indicating  the  presence  of  bacteria 
more  nearly  related  to  pathogenic  types.  The  com- 
parative value  of  the  two  methods  has  been  well  dis- 
cussed by  Whipple  (1913).  The  use  of  gelatin  is  not 
only  more  time-consuming,  but  requires  the  use  of  a 


42         ELEMENTS  OF  WATER  BACTERIOLOGY 

special  2o-degree  incubator  which  is  difficult  to  regulate. 
The  37-degree  incubator  must  be  provided  in  any  case 
for  the  isolation  of  B.  coli.  On  the  other  hand,  the 
time  seems  hardly  ripe  for  the  abandonment  of  the 
2o-degree  count,  which  has  been  used  for  20  years  all 
over  the  civilized  world,  and  for  the  interpretation  of 
which  we  have  very  complete  data.  There  is  at  present 
no  such  sound  basis  for  interpreting  the  37-degree 
count,  and  in  many  cases,  as  in  the  control  of  water 
nitration  plants,  the  37-degree  numbers  are  too  small 
to  be  of  any  practical  value.  Furthermore  the  20- 
degree  count  may  furnish  evidence  of  surface  con- 
tamination as  distinguished  from  fecal  pollution,  which 
is  often  of  considerable  value. 

The  authors  have  always  urged  the  use  of  the  37- 
degree  count  along  with  the  2o-degree  count  as  furnish- 
ing most  valuable  information;  but  this  is  very  different 
from  the  substitution  of  one  count  for  the  other. 
The  recommendation  that  the  2o-degree  count  be 
abandoned,  with  no  evidence  to  warrant  such  a  revolu- 
tionary change,  and  no  experimental  results  on  which 
to  base  an  interpretation  of  the  37-degree  count,  has 
aroused  vigorous  opposition  from  a  large  majority 
of  practical  water  bacteriologists.  At  the  Washington 
meeting  of  the  American  Public  Health  Association 
in  September,  1912,  it  was  resolved  "  that  in  the  opinion 
of  the  Laboratory  Section  of  the  American  Public 
Health  Association,  ordinary  routine  examination  of 
water  for  sanitary  purposes,  and  in  the  control  of 
purification  plants,  for  the  present  should  include  the 
determination  of  the  number  of  bacteria  developing 


QUANTITATIVE  EXAMINATION  OF  WATER     43 

at  20  degrees  and  at  37  degrees  and  a  presumptive 
test  for  B.  coli  in  lactose  bile." 

This  action  of  the  section  responsible  for  the  appoint- 
ment of  the  Standard  Methods  Committee  appears  to 
supersede  the  report  of  the  committee  itself  and  makes 
the  combination  of  the  20-  and  37-degree  counts  the 
standard  American  procedure.  The  2o-degree  count 
may  be  made  on  either  gelatin  or  agar;  but  it  is  the 
2o-degree  count  which  will  be  discussed  in  this  chapter, 
leaving  the  body  temperature  count  for  consideration 
in  Chapter  IV. 

The  exact  composition  of  the  medium  is,  of  course, 
of  prime  importance  in  controlling  the  number  of 
bacteria  which  will  develop.  The  figures  previously 
cited  in  connection  with  the  discussion  of  Hesse's 
Nahrstoff  agar  show  how  bacterial  counts  may  vary 
with  media  of  widely  different  composition.  The 
table  quoted  on  page  44  from  Gage  and  Phelps  (1902), 
shows  the  considerable  differences  which  may  be  due 
to  the  presence  or  absence  of  meat  infusion,  peptone, 
etc.,  in  media  of  generally  similar  character  (compare 
the  figures  for  plain  gelatin,  peptone,  gelatin,  and  meat 
gelatin).  Much  slighter  variations  than  this,  however, 
are  significant.  The  reaction  of  the  medium  was  found 
as  early  as  1891  to  be  important,  for  Reinsch  (Reinsch, 
1891)  showed  in  that  year  that  the  addition  of  one 
one-hundredth  of  a  gram  of  sodium  carbonate  to  the 
liter  increased  sixfold  the  number  of  bacteria  develop- 
ing. Fuller  (Fuller,  1895)  and  Sedgwick  and  one  of 
us  (Sedgwick  and  Fresco tt,  1895),  working  indepen- 
dently, established  the  fact  that  an  optimum  reaction 


44          ELEMENTS  OF  WATER  BACTERIOLOGY 


existed  for  most  water  bacteria  and  that  a  devia- 
tion either  way  decreased  the  number  of  colonies 
developing. 

TABLE    SHOWING    PERCENTAGES    OF   BACTERIA   DEVEL- 
OPING ON  MEDIA  OF  DIFFERENT  COMPOSITIONS 

(GAGE  AND  PHELPS,  1902) 


Medium. 

Days' 

Cour 

t. 

2 

3 

4 

5 

6 

7 

8 

9 

Nahrstoff  agar 

IQ 

60 

78 

8* 

0  ^ 

QO 

OQ 

IOO 

Nahrstoff  peptone  agar  
Peptone  agar  
Meat  agar 

10 

II 
8 

22 

16 
13 

26 

22 

16 

28 
23 

30 

24 

I  7 

30 
24 

I  7 

3° 

24 
1  7 

30 
24 

I  ^ 

Plain  agar.    ... 

8 

10 

I? 

14 

14 

14 

14 

14 

Regular  agar  

7 

g 

II 

II 

1  1 

II 

II 

1  1 

Nahrstoff  glycerin  agar  
Nahrstoff  meat  agar 

6 

7 

10 

7 

II 

8 

II 

8 

II 
IO 

II 
IO 

II 
IO 

II 
IO 

Meat  gelatin  

12 

IQ 

24 

06 

26 

06 

26 

26 

Peptone  gelatine  

7 

12 

18 

20 

20 

2O 

20 

2O 

Standard  gelatin    . 

8 

IO 

ii 

12 

17 

I  3 

I  3 

I  3 

Plain  gelatin  

i 

6 

12 

12 

I  ^ 

I?, 

13 

13 

Nahrstoff  gelatin  

5 

6 

g 

11 

13 

13 

12 

13 

Whipple  (Whipple,  1902)  has  shown  that  not  only 
the  particular  kind  of  gelatin  used,  but  its  exact  physical 
condition  as  affected  by  sterilization  and  other  previous 
treatments,  will  materially  affect  the  results  obtained. 
Gage  and  Adams  (1904)  found  marked  differences  in 
counts  as  the  result  of  the  use  of  the  two  best-known 
commercial  peptones.  A  long  series  of  waters  plated 
on  agar  made  up  with  Merck's  and  Witte's  peptones, 
respectively,  showed  the  average  relative  results  in  the 
table  on  page  45. 


QUANTITATIVE  EXAMINATION  OF  WATER     45 

AVERAGE    RELATIVE   NUMBER   OF   BACTERIA    ON   PEP- 
TONE AGAR  WITH  DIFFERENT  PEPTONES 

(GAGE  AND  ADAMS,  1904) 


DAYS 

2 

4 

6 

8 

10 

12 

Merck's 

•2-2 

ci 

67 

80 

08 

Witte's          

38 

r? 

IOO 

JOO 

IOO 

IOO 

The  same  authors  showed  that  the  composition  of 
the  water  used  exercised  a  marked  selective  action  upon 
the  development  of  bacteria.  Agar  made  up  with 
sewage  permitted  a  maximum  growth  of  sewage  bacteria 
and  showed  no  colonies  when  inoculated  with  filtered 
city  water.  On  the  other  hand  agar  made  up  with  city 
water  showed  100  per  cent  of  the  bacteria  present  in 
city  water  and  river  water,  three-quarters  of  those 
present  in  sewage  and  less  than  half  of  those  present 
in  sewage  effluents. 

Hesse  (1904)  found  that  the  number  of  bacteria 
developing  on  Nahrstoff  agar  varied  with  the  composi- 
tion of  the  glass  tubes  in  which  the  media  had  previously 
been  sterilized.  The  more  soluble  glasses  yielded 
sufficient  alkali  to  the  medium  to  inhibit  four-fifths  of 
the  bacteria  present  in  certain  cases. 

All  these  facts  make  it  evident  that  only  the  strictest 
adherence  to  a  standard  method  can  ensure  comparable 
results;  the  ordinary  nutrient  gelatin  or  agar  should 
then  in  all  practical  sanitary  work  be  made  up  from 
distilled  water,  meat  infusion,  peptone  and  gelatin  or 
agar,  in  exact  accordance  with  the  directions  of  the 
Standard  Methods  Committee. 


46         ELEMENTS  OF  WATER  BACTERIOLOGY 

Even  the  standard  procedure  fails  to  ensure  uniformity 
in  one  important  respect.  The  meat  infusion  which  it 
calls  for  is  in  itself  a  highly  variable  quantity.  Gage 
and  Adams  (1904),  in  the  examination  of  fifteen  lots 
of  beef  infusion,  found  variations  of  nearly  i  per  cent 
in  organic  solids  (calculated  on  the  weight  of  the  whole 
infusions),  after  the  final  filtration.  The  organic 
constituents  of  the  meat  infusion  varied,  therefore, 
among  themselves  by  nearly  the  total  amount  of  pep- 
tone added.  It  is  to  be  hoped  that  the  standard  methods 
may  soon  be  so  revised  as  to  eliminate  this  necessarily 
uncertain  constituent  of  nutrient  media.  Criticisms 
of  detail  must,  however,  give  way  to  the  importance 
of  securing  fairly  comparable  results;  and  the  con- 
fusion which  would  follow  the  use  by  individual  bac- 
teriologists of  media  made  without  meat  would  out- 
balance the  errors  inherent  in  the  standard  procedure. 

Incubation.  Incubation  should  take  place  in  a  dark, 
well-ventilated  chamber  where  the  temperature  is 
kept  substantially  constant  at  20  degrees  and  where 
the  atmosphere  is  practically  saturated  with  moisture. 
It  has  been  shown  by  Whipple  (Whipple,  1899)  and  others 
that  the  number  of  bacteria  developing  in  plate  cultures 
is  to  a  certain  extent  dependent  upon  the  presence  of 
abundant  oxygen  and  moisture.  Thus,  reckoning  the 
number  of  bacteria  developing  in  a  moist  chamber  at 
100,  the  percentage  counts  obtained  in  an  ordinary 
incubator  were  as  follows:  75  when  the  relative  humid- 
ity of  the  incubator  was  60  per  cent  of  saturation;  82 
when  it  was  75  per  cent;  98  when  it  was  95  per  cent. 
This  source  of  error  may  be  avoided  by  the  use  of  ven- 


QUANTITATIVE  EXAMINATION  OF  WATER      47 


tilated  dishes  and  by  the  presence  of  a  pan  of  water  in 
the  incubating  chamber. 

According  to  American  and  German  practice,  plates 
made  for  sanitary  water  analysis  are  counted  at  the 
end  of  48  hours.  The  English  Committee  appointed 
to  consider  the  standardization  of  methods  for  the 
Bacterioscopic  Examination  of  Water  (1904)  fixed  the 
time  at  72  hours.  French  bacteriologists,  and  some 
Germans  (Hesse  and  Niedner,  1906),  still  recommend 
longer  periods,  and  the  following  table  from  Miquel 
and  Cambier  (Miquel  and  Cambier,  1902)  shows  that 
many  bacteria  fail  to  appear  in  our  ordinary  procedure. 
It  is,  however,  in  the  main,  the  characteristic  water 
bacteria  which  develop  slowly,  sewage  bacteria  almost 
without  exception  being  rapid  growers.  The  longer 
period  of  incubation  is,  therefore,  not  only  inconvenient, 
but  undesirable,  since  it  obscures  the  difference  between 
good  and  bad  waters. 

EFFECT  OF  THE  LENGTH  OF  INCUBATION  OF  WATER 
BACTERIA  IN  GELATIN  UPON  THE  NUMBER  OF 
COLONIES  DEVELOPING 

(MIQUEL  AND  CAMBIER,  1902) 


Length  of  Incubation. 

Colonies 
Developed. 

Length  of  Incubation. 

Colonies 
Developed. 

I  day 

2O 

o  days 

821 

2  days  

136 

10  days   .  . 

8qo 

7  days 

2  CA 

1  1  days 

802 

4  days.  .  . 

187 

12  days 

Q2I 

5  days  

S^o 

i  3  days  .  . 

QCI 

6  days    . 

6*7 

Mdays 

076 

7  days  

72CJ 

i  ^  days  .  . 

IOOO 

8  days    .  . 

780 

48         ELEMENTS  OF  WATER  BACTERIOLOGY 

Counting.  The  number  of  bacteria  is  determined  by 
counting  the  colonies  developed  upon  the  plate,  with  the 
aid  of  a  lens  magnifying  at  least  five  diameters.  For 
convenience  in  counting  the  plate  may  be  placed  upon 
a  glass  plate  ruled  in  centimeter  squares  and  set  over  a 
black  tile;  or  the  tile  itself  may  be  ruled.  As  has 
already  been  said,  it  is  desirable  that  the  number  of 
colonies  should  not  exceed  200,  for  when  the  number 
is  very  high  the  colonies  grow  only  to  a  small  size, 
making  counting  laborious  and  inaccurate,  and  many 
do  not  develop  at  all.  The  best  results  are  obtained 
with  numbers  ranging  from  50  to  200. 

When  it  is  possible  to  do  so,  all  the  colonies  on  the 
plate  should  be  counted.  When  they  exceed  400  or 
500  it  is  often  easier,  and  fully  as  accurate,  to  count 
a  fractional  part  of  the  plate  and  estimate  the  total 
number  therefrom.  This  should  not  be  done,  however, 
except  in  case  of  necessity. 

Ayers  (1911)  has  suggested  two  counting  devices 
which  will  be  found  very  useful  where  a  great  many 
plates  have  to  be  handled.  For  getting  the  best  possible 
transmitted  light,  he  places  his  plate  on  the  ground- 
glass  top  of  a  wooden  box,  7  inches  square,  with  one  side 
open  to  admit  light,  which  is  reflected  upward  by  a 
plane  mirror  set  in  the  box  at  an  angle  of  45  degrees. 
An  ordinary  graduated-glass  counting  plate  may  be 
placed  between  the  ground-glass  and  the  Petri  dish,  and 
the  eyes  are  protected  from  direct  light  by  a  screen 
rising  from  the  open  side  of  the  box.  For  picking 
colonies  from  a  gelatin  plate  in  a  warm  room,  he  places 
between  the  ground  glass  and  the  Petri  dish  a  copper 


QUANTITATIVE  EXAMINATION  OF  WATER      49 


box  with  top  and  bottom  of  glass  7  inches  square  and 
ij  inches  deep,  through  which  cold  water  is  allowed  to 
circulate. 

Expression  of  Quantitative  Results.  It  is  customary 
in  determining  numbers  to  make  plates  in  duplicate, 
thereby  affording  a  check  upon  one's  own  work.  Owing 
to  the  lack  of  precision  in  the  method,  the  limit  of 
experimental  error  is  a  wide  one.  It  should  be  possible 
for  careful  manipulators  to  obtain  results  within  10 
per  cent  of  each  other,  but  a  closer  agreement  than  this 
is  hardly  to  be  expected.  It  has  been  suggested  by  the 
committee  of  the  American  Public  Health  Association 
that  the  following  mode  of  expressing  results  be  adopted 
in  order  to  avoid  the  appearance  of  a  degree  of  accuracy 
which  the  methods  do  not  warrant. 

NUMBERS  OF  BACTERIA  FROM 


1-50  shall  be  recorded  to  the 

51-100 

101-250 

251-500 

501-1000 

1001-10,000 

10,001-50,000 

50,001-100,000 

100,001-500,000 

500,001-1 ,000,000 

i  ,000,001-5,000,000 


nearest  unit 

5 
10 

25 
50 

IOO 

500 

1,000 
10,000 
50,000 

100,000 


The  determination  of  numbers  of  bacteria  in  water 
in  the  field  has  frequently  been  attempted.  Since 
the  laboratory  method  of  "plating  out"  is  difficult 
to  use  in  field  work,  the  Esmarch  tube  process  has  often 
been  employed.  This  consists  in  introducing  into  a 
tube  of  melted  gelatin  or  agar  i  c.c.  of  the  water  and  then 


50         ELEMENTS  OF  WATER  BACTERIOLOGY 

rotating  the  tube  until  the  medium  has  solidified  in  a 
thin  layer  on  the  inner  wall.  Other  bacteriologists 
have  devised  ingenious  field  kits  for  adapting  the  plate 
method  to  this  purpose,  of  which  one  very  good  form 
has  recently  been  described  by  Van  Buskirk  (1912). 
The  opportunity  for  air  infection  in  work  done  outside 
a  proper  laboratory  is,  however,  always  great;  and  it 
is  almost  impossible  to  secure  proper  conditions  for 
incubation  in  any  makeshift  establishment.  On  the 
whole,  the  authors  are  of  the  opinion  that  laboratory 
examinations  are  to  be  preferred  to  those  made  in  the 
field,  if  a  laboratory  can  be  reached  within  12  hours 
or  so  of  the  time  of  collection  of  the  samples. 


CHAPTER  III 

THE  INTERPRETATION  OF  THE  QUANTITATIVE 
BACTERIOLOGICAL  EXAMINATION 

Standards  for  Potable  Water.  The  information  fur- 
nished by  quantitative  bacteriology  as  to  the  antecedents 
of  a  water  is  in  the  nature  of  circumstantial  evidence 
and  requires  judicial  interpretation.  No  absolute  stand- 
ards of  purity  can  be  established  which  shall  rigidly 
separate  the  good  from  the  bad.  In  this  respect  the 
terms  "  test  "  and  "  analysis  "  so  universally  used 
are  in  a  sense  inappropriate.  Some  scientific  problems 
are  so  simple  that  they  can  be  definitely  settled  by  a 
test.  The  tensile  strength  of  a  given  steel  bar,  for 
example,  is  a  property  which  can  be  determined. 
In  sanitary  water  examination,  however,  the  factors 
involved  are  so  complex,  and  the  evidence  neces- 
sarily so  indirect,  that  the  process  of  reasoning  much 
more  resembles  a  doctor's  diagnosis  than  an  engineering 
test. 

The  older  experimenters  attempted  to  establish 
arbitrary  standards,  by  which  the  sanitary  quality  of  a 
water  could  be  fixed  automatically  by  the  number  of 
germs  alone.  Thus  Miquel  (Miquel,  1891)  published  a 
table  according  to  which  water  with  less  than  10  bac- 
teria per  c.c.  was  "  excessively  pure,"  with  10  to  100 

51 


52         ELEMENTS  OF  WATER  BACTERIOLOGY 

bacteria,  "  very  pure/'  with  100  to  1000  bacteria, 
"  pure/'  with  1000  to  10,000  bacteria,  "  mediocre,"  with 
10,000  to  100,000  bacteria,  "  impure,"  and  with  over 
100,000  bacteria,  "  very  impure."  Few  sanitarians 
would  care  to  dispute  the  appropriateness  of  the  titles 
applied  to  waters  of  the  last  two  classes;  but  many  bac- 
teriologists have  placed  the  standard  of  "  purity  "  much 
lower.  The  limits  set  by  various  German  observers 
range,  for  example,  from  50  to  300.  Dr.  Sternberg 
(Sternberg,  1892)  in  a  more  conservative  fashion, 
has  stated  that  a  water  containing  less  than  100  bacteria, 
is  presumably  from  a  deep  source  and  uncontaminated 
by  surface  drainage;  that  one  with  500  bacteria  is 
open  to  suspicion;  and  that  one  with  over  1000  bac- 
teria is  presumably  contaminated  by  sewage  or  surface 
drainage.  This  is  probably  as  satisfactory  an  arbitrary 
standard  as  could  be  devised,  but  any  such  standard 
must  be  applied  with  great  caution.  The  source  of 
the  sample  is  of  vital  importance  in  the  interpretation 
of  analyses;  a  bacterial  count  which  would  condemn 
a  spring  might  be  quite  normal  for  a  river;  only  figures 
in  excess  of  those  common  to  unpolluted  waters  of  the 
same  character  give  an  indication  of  danger.  Fur- 
thermore, the  bacteriological  tests  are  far  more  delicate 
than  any  others  at  our  command,  very  minute  addi- 
tions of  food  material  causing  an  immense  multiplica- 
tion of  the  microscopic  flora.  This  delicacy  necessarily 
requires,  both  in  the  process  of  analysis  and  the  inter- 
pretation of  results,  a  high  degree  of  caution.  As 
pointed  out  in  the  previous  chapter,  the  touch  of  a 
finger  or  the  entrance  of  a  particle  of  dust  may  wholly 


QUANTITATIVE  BACTERIOLOGICAL  ANALYSIS    53 

destroy  the  accuracy  of  an  examination.  Even  the 
slight  disturbance  of  conditions  incident  upon  the 
storage  of  a  sample  after  it  has  been  taken  may  in  a 
few  hours  wholly  alter  the  relations  of  the  contained 
microbic  life.  It  is  necessary,  then,  in  the  first  place, 
to  exercise  the  greatest  care  in  allowing  for  possible 
error  in  the  collection  and  handling  of  bacteriological 
samples;  and  in  the  second  place,  only  well-marked 
differences  in  numbers  should  be  considered  significant. 

In  the  early  days  of  the  science,  discussion  ran  high 
as  to  the  interpretation  of  bacteriological  analysis; 
and  particularly  as  to  the  relation  of  bacterial  numbers 
to  the  organic  matter  present  in  a  water.  Different 
observers  obtained  inconsistent  results,  and  Bolton 
(Bolton,  1886)  concluded  that  there  was  no  relation 
whatever  between  the  organic  pollution  of  a  water  and 
its  bacterial  content.  Tiemann  and  Gartner  (Tiemann 
and  Gartner,  1889)  furnished  the  key  to  the  difficulty 
in  their  statement  that  there  are  two  classes  of  bacteria, 
the  great  majority  of  species  normally  occurring  in 
the  earth  or  in  decomposing  organic  matter,  which 
require  abundance  of  nutriment,  and  certain  peculiar 
water  bacteria  which  can  multiply  in  the  presence  of 
such  minute  traces  of  ammonia  as  are  present  in  ordi- 
nary distilled  water.  Even  these  prototrophic  or 
semi-prototrophic  forms,  however,  require  a  definite 
amount  of  food  of  their  own  kind. 

Kohn  (1906)  determined  the  minimal  nutrient  mate- 
rial requisite  for  certain  of  them  and  found  that  they 
could  develop  in  the  presence  of  198 X  io~10  to  I98X  io"13 
per  cent  of  dextrose,  66Xio~13  to  66Xio"17  per  cent 


54         ELEMENTS  OF  WATER  BACTERIOLOGY 

ammonium  sulphate  and  66Xio~13  to  66Xio~19  per 
cent  ammonium  phosphate.  Similar  minute  amounts 
of  organic  matter  are  found  in  the  purest  of  natural 
waters  and  under  exceptional  conditions  certain  species 
of  bacteria  may  therefore  multiply  in  bottled  samples, 
or,  at  times,  in  a  well  or  the  basin  of  a  spring.  In 
normal  surface-waters,  such  growths  of  the  prototrophic 
forms  do  not  apparently  occur.  Here  it  is  found  as  a 
matter  of  practical  experience  that  the  number  of  bac- 
teria present  depends  upon  the  extent  to  which  the 
water  has  been  contaminated  with  decomposing  organic 
matter,  either  by  pollution  with  sewage  or  by  contact 
with  the  surface  of  the  ground.  The  bacterial  content 
varies  as  the  extent  and  character  of  the  contamination 
varies.  It  measures  not  merely  organic  matter,  but 
organic  matter  in  a  state  of  active  decay,  and  like  the 
ammonias  and  other  features  of  the  sanitary  chemical 
analysis,  indicates  fresh  organic  pollution,  with  the  added 
advantage  that  the  presence  of  the  stable  nitrogenous 
compounds  often  present  in  peaty  waters  introduces 
no  error  in  the  bacteriological  analysis. 

Bacterial  Content  of  Surface-waters.  In  judging  of  a 
surface-water  the  student  will  be  aided  by  reference  to 
the  figures  given  for  certain  normal  sources  in  Chapter 
I;  the  Boston  tap  water  with  50  to  200  bacteria  per 
c.c.  (Philbrick,  1905)  and  the  water  of  Lake  Zurich 
with  an  average  of  71  in  summer  and  184  in  winter 
(Cramer,  1885)  may  be  taken  as  typical  of  good  potable 
waters;  and  numbers  much  higher  than  these  are  open 
to  suspicion,  since  all  contamination  whether  contributed , 
by  sewage  or  by  washings  from  the  surface  of  the 


QUANTITATIVE  BACTERIOLOGICAL  ANALYSIS    55 

ground  is  a  possible  source  of  danger.  The  excess  of 
bacteria  in  surface-waters  during  the  spring  and  winter 
months  is  by  no  means  an  exception  to  the  general 
rule  that  high  numbers  are  significant,  since  the  peril 
from  supplies  of  this  character  is  clearly  shown  by  the 
spring  epidemics  of  typhoid  fever  which  at  the  times  of 
melting  snow  visit  communities  making  use  of  unpro- 
tected surface-waters.  Streams  receiving  direct  con- 
tributions of  sewage  exhibit  a  similar  excess  of  bacteria 
at  all  times,  numbers  rising  to  an  extraordinary  height 
near  the  point  of  pollution  and  falling  off  below  as  the 
stream  suffers  dilution  and  the  sewage  organisms  perish. 
Miquel  (Miquel,  1886)  records  300  bacteria  per  c.c. 
in  the  water  of  the  Seine  at  Choisy,  above  Paris;  1200 
at  Bercy  in  the  vicinity  of  the  city,  and  200,000  at  St. 
Denis  after  the  entrance  of  the  drainage  of  Paris. 
Prausnitz  (Prausnitz,  1890)  found  531  bacteria  per  c.c. 
in  the  Isar  above  Munich,  227,369  near  the  entrance  of 
the  principal  sewer,  9111  at  a  place  13  kilometers 
below  the  city,  and  2378  at  Freising,  20  kilometers 
further  down.  Jordan  (Jordan,  1900),  in  his  study 
of  the  fate  of  the  sewage  of  Chicago,  found  1,245,000 
bacteria  per  c.c.  in  the  drainage  canal  at  Bridgeport, 
650,000  at  Lockport,  29  miles  below,  and  numbers 
steadily  decreasing  to  3660  at  Averyville,  159  miles 
below  the  point  of  original  pollution.  Below  Avery- 
ville the  sewage  of  Peoria  enters  and  the  numbers 
rise  to  758,000  at  Wesley  City,  decreasing  to  4800  in 
123  miles  flow  to  Kampsville.  Brezina  (1906)  found 
1900  bacteria  per  c.c.  in  the  Danube  River  above,  and 
1 10,000  at  the  north  of  the  Danube  canal.  This  number 


56         ELEMENTS  OF  WATER  BACTERIOLOGY 

fell  to  85,000  one  kilometer  below,  62,000  four  kilo- 
meters below,  and  40,000  seven  kilometers  down  the 
stream.  Vincent  (1905)  records  from  1000  to  46,000 
bacteria  per  c.c.  in  the  waters  of  more  or  less  polluted 
French  rivers.  Mayer  (1902),  on  the  other  side  of  the 
world,  found  21  and  35  bacteria  per  c.c.  in  the  Shaho 
River,  near  its  source,  in  the  vicinity  of  the  great 
Chinese  Wall  and  from  100,000  to  600,000  in  the  highly 
polluted  Whangpo  near  its  mouth. 

Bacterial  Content  of  Ground-waters.  In  ground- 
waters  we  have  seen  that  bacteria  may  occasionally 
be  present  in  considerable  numbers,  but  if  so  they  are 
generally  organisms  of  a  peculiar  character,  incapable 
of  development  on  the  ordinary  nutrient  media  in  the 
standard  time.  Thus  in  48  hours  we  often  obtain 
counts  measured  only  in  units  or  tens  such  as  have 
been  recorded  in  Chapter  I.  When  higher  numbers 
are  present,  the  general  character  of  the  colonies  must 
be  taken  into  account,  since  besides  the  slowly-growing 
forms  certain  other,  water  bacteria,  which  require  a 
comparatively  small  amount  of  nutriment,  may  multiply 
at  times  in  a  deep  well  or  the  basin  of  a  spring.  In 
such  a  case,  however,  the  appearance  of  the  plates  at 
once  reveals  the  peculiar  conditions,  for  the  colonies 
are  of  one  kind  and  that  distinct  from  any  of  the  sewage 
species.  Thus  Dunham  (Dunham,  1889)  reports  that  the 
mixed  water  from  a  series  of  driven  wells  gave  2  bacteria 
per  c.c.,  while  another  well,  situated  just  like  the  others, 
contained  5000,  all  belonging  to  a  single  species  common 
in  the  air.  Except  in  such  peculiar  cases  as  this  high 
numbers  in  a  ground-water  mean  contamination. 


QUANTITATIVE  BACTERIOLOGICAL  ANALYSIS    57 

Bacteria  in  Filtered  Waters.  The  process  of  slow 
sand  filtration  for  the  purification  of  unprotected 
surface-water  is  essentially  similar  to  the  action  which 
takes  place  in  nature  when  rain  soaks  through  the 
ground  to  appear  in  wells  and  springs;  and  it  is  in  the 
examination  of  the  effluent  from  such  municipal  plants 
that  the  quantitative  bacteriological  analysis  finds, 
perhaps,  its  most  important  application.  The  chemical 
changes  which  occur  in  the  passage  of  water  through 
sand  at  a  rate  of  1,000,000  or  2,000,000  gallons  per  acre 
per  day  are  so  slight  as  to  be  negligible.  The  bacteria 
present  should,  however,  suffer  a  reduction  of  98  or 
99  per  cent,  and  their  numbers  furnish  the  best  standard 
for  measuring  the  efficiency  of  such  filtration  plants. 
At  Lawrence,  in  1905,  Clark  found  an  average  of  12,700 
bacteria  per  c.c.  in  the  raw  water  of  the  Merrimac 
River,  while  the  number  present  in  the  filtered  water 
was  only  70  (Massachusetts  State  Board  of  Health, 
1906).  Where  the  number  of  bacteria  in  the  applied 
water  is  smaller  it  is  difficult  to  obtain  so  high  a  per- 
centage efficiency.  At  Washington,  for  example,  pro- 
longed sedimentation  generally  reduces  the  bacterial 
numbers  to  less  than  a  thousand  and  it  is  almost  impos- 
sible to  secure  a  99  per  cent  removal.  The  actual 
numbers  of  bacteria  in  the  effluent  are,  however,  much 
lower  than  at  Lawrence.  The  monthly  average  results 
obtained  for  a  year  at  these  two  plants  are  tabulated 
on  page  58. 

Mechanical  filtration  gives  similar  results.  Fuller 
at  Cincinnati  (Fuller,  1899)  records  27,200  organisms 
per  c.c.  in  the  water  of  the  Ohio  River  between 


58 


ELEMENTS  OF  WATER  BACTERIOLOGY 


September  21,  1898,  and  January  25,  1899,  while 
the  average  content  of  the  effluent  from  the  Jewell 
filter  was  400.  Data  with  regard  to  the  operation  of 
mechanical  niters  are  now  abundant,  since  all  over  the 
world  the  operation  of  these  plants  is  controlled  by 
bacteriological  methods.  Recently  Johnson  (1907)  has 
reported  some  interesting  results  from  the  far  East. 
At  Osaka,  Japan,  an  average  of  200  bacteria  per  c.c.  in 
the  raw  water  of  the  Yodo  River  was  reduced,  in  1905, 
to  an  average  of  25  by  slow  sand  niters;  at  Bethmangala, 
India,  in  1906,  mechanical  niters  treated  the  water  of  the 
'Palar  River,  containing  4350  bacteria  per  c.c.,  and 
yielded  an  effluent  with  only  13  per  c.c.  (Johnson,  1907). 
The  average  monthly  results  obtained  with  the  new 
mechanical  filter  plant  at  Harrisburg,  Pa.,  are  included 
in  the  table  below  for  comparison  with  the  figures 

REMOVAL  OF  BACTERIA  BY  NATURAL  SAND  FILTERS 
AND  MECHANICAL  FILTERS.  BACTERIA  PER  C.C.  IN 
APPLIED  WATER  AND  EFFLUENT.  MONTHLY  AVER- 
AGES 


Month. 

Washington,  1906. 

Lawrence,  1905. 

Harrisburg,  1906. 

Applied 
Water. 

Effluent. 

Applied 
Water. 

Effluent. 

Applied 
Water. 

Effluent. 

January  .... 

1500 

39 

14,200 

no 

9,510 

104 

February  .  .  . 

550 

16 

14,800 

55 

21,228 

298 

March  

650 

19 

10,300 

55 

3^326 

75 

April  

400 

22 

3,600 

170 

39-905 

42 

May 

65 

17 

1,900 

12 

6,187 

86 

June  

o 
22O 

17 

9,600 

9 

2,903 

3i 

July  

1  60 

26 

3^0° 

55 

685 

10 

August  

190 

14             19,500 

37 

1,637 

5 

September  .  . 

I30 

14 

13,500 

44 

836     |            12 

October  

275 

16 

39,800 

110 

7-575 

63 

November  .  . 

22O 

12 

8.700 

70 

26,224 

236 

December..  .  . 

700 

45 

•  •       !    37,525 

163 

QUANTITATIVE  BACTERIOLOGICAL  ANALYSIS    59 

recorded  at  Washington  and  Lawrence;  and  these 
may  be  taken  as  typical,  since  the  Harrisburg  plant  is 
the  latest  of  its  type,  as  the  Washington  plant  is  the 
newest  and  most  perfectly  equipped  of  slow  sand 
niters. 

In  well-managed  purification  plants  the  bacteria 
in  the  effluent  are  determined  daily,  and  any  deviation 
from  the  normal  value  at  once  reveals  disturbing  factors 
which  may  impair  the  efficiency  of  the  process.  In 
Prussia  official  regulations  demand  such  systematic 
examinations  and  prescribe  50  as  the  maximum  number 
of  bacteria  allowable  in  the  filtered  water.  In  the 
same  way  the  condition  of  an  unpurified  surface  supply 
may  be  determined  by  daily  bacteriological  analyses 
and  warnings  of  danger  issued  to  the  public,  as  has 
been  done  at  Chicago  and  other  cities.  In  general, 
any  regular  determination  of  variations  from  a  normal 
standard  furnishes  ideal  conditions  for  the  bacteriological 
methods;  and  the  detection  by  Shuttleworth  (Shuttle- 
worth,  1895)  of  a  break  in  a  conduit  under  Lake  Ontario 
by  a  rise  in  the  bacteria  of  the  Toronto  water-supply 
may  be  cited  as  a  classic  example  of  its  application. 

Often,  however,  the  expert  is  called  to  pass  upon  the 
character  of  a  water  of  which  no  series  of  analyses  is 
available.  In  such  cases  an  inspection  of  the  location 
from  which  the  water  comes  should  be  insisted  on,  as  a 
sound  interpretation  of  a  water  analysis  can  only  be  made 
with  a  reasonably  full  knowledge  of  the  source  of  the 
sample.  After  a  careful  sanitary  inspection,  however, 
the  comparison  of  the  result  of  even  a  single  examina- 
tion with  the  normal  range  for  waters  of  the  same  class 


60         ELEMENTS  OF  WATER  BACTERIOLOGY 

may  prove   of   great   significance,   as   a   few   practical 
examples  may  make  clear  (Winslow,  1901). 

In  the  spring  of  1900  the  city  of  Hartford,  Conn., 
was  using  a  double  supply,  from  the  Connecticut  River 
and  from  a  series  of  impounding  reservoirs  among  the 
hills.  A  single  series  of  plates  showed  from  4000  to 
7000  bacteria  per  c.c.  in  the  water  of  the  river,  while 
the  reservoir  water  contained  300  to  900.  The  abandon- 
ment of  the  river  supply  followed,  and  at  once  the 
excessive  amount  of  typhoid  fever  in  the  city  was 
curtailed. 

In  the  fall  of  1900,  Newport,  R.  I.,  experienced  an 
outbreak  of  typhoid  fever,  and  when  suspicion  was 
thrown  upon  the  surface  water-supply,  chemical  analysis 
of  the  latter  was  not  wholly  reassuring;  but  there  were 
only  334  bacteria  per  c.c.  in  the  water  from  the  taps, 
while  a  well  in  the  infected  district  gave  6100.  It  was 
no  surprise  to  find,  on  a  further  study  of  the  epidemic, 
that  the  well  was  largely  at  fault  and  the  public  supply 
was  not. 

In  the  case  of  ground-water  the  evidence  is  usually 
even  more  distinct.  At  Framingham,  Mass.,  in  1903, 
high  chlorin  content  in  the  public  supply,  drawn  from  a 
filter  gallery  beside  a  lake,  had  led  to  public  anxiety. 
Five  samples  from  different  parts  of  the  system  showed 
averages  of  i,  2,  2,  2,  and  4  bacteria  per  c.c.;  and 
taking  this  in  conjunction  with  the  other  features  of 
the  bacteriological  analysis,  it  was  possible  to  report 
that  any  pollution  introduced  upon  the  gathering 
ground  had  at  the  time  of  examination  been  entirely 
removed. 


CHAPTER  IV 

DETERMINATION     OF     THE     NUMBER     OF     ORGANISMS 
DEVELOPING   AT   THE   BODY  TEMPERATURE 

Relation  between  Counts  Made  at  20°  and  37°.    The 

count  of  colonies  upon  the  gelatin  plate  measures, 
as  we  have  pointed  out,  the  number  of  the  metatrophic 
bacteria  in  general;  and  the  distribution  of  these  forms 
corresponds  with  the  decomposition  of  organic  matter 
wherever  it  may  occur.  In  this  great  class,  there  are 
some  species  which  will  grow  under  a  wide  variety  of 
conditions.  These  are  present  in  most  waters  in  small 
numbers,  and  in  sources  contaminated  with  wash  from 
decaying  vegetable  matter  they  occur  in  abundance. 
Other  metatrophic  forms,  however,  through  a  semi- 
parasitic  mode  of  life,  have  become  specially  adapted 
to  the  peculiar  conditions  characteristic  of  the  animal 
body;  and  these  bacteria  possess  the  property  of  develop- 
ing most  actively  at  the  temperature  of  the  human 
body,  37°  C.,  which  altogether  checks  the  growth  of 
the  majority  of  normal  earth  and  water  forms.  The 
determination  of  the  number  of  organisms  growing 
at  the  body  temperature  may  throw  light,  then,  on  the 
presence  of  direct  sewage  pollution,  since  the  bacteria 
from  the  alimentary  canal  flourish  under  such  con- 
ditions, while  most  of  those  derived  from  other  sources 
do  not.  Savage  classifies  the  bacteria  which  may 

61 


62         ELEMENTS  OF  WATER  BACTERIOLOGY 

be  found  in  water  under  three  headings:  normal  inhab- 
itants, like  B.  fluorescens;  unobjectionable  aliens 
(from  soil),  like  B.  mycoides,  and  objectionable  aliens 
(from  excreta),  like  B.  coli.  The  first  sort  and  many 
of  the  second  sort  are  generally  unable  to  grow  at  37 
degrees.  This  criterion  is  not  an  absolute  one.  Savage, 
(1906)  reports  an  experiment  in  which  unpolluted  soil, 
which  had  not  been  manured  or  cultivated  for  at  least 
3  years,  was  added  to  tap  water,  with  the  result  that  a 
20°  count  of  76  was  increased  to  1970,  and  a  37°  count 
of  3  was  raised  to  1630.  In  this  case  most  of  the 
bacteria  in  the  soil  were  capable  of  development  at 
body  temperature.  Experience  shows,  however,  that 
the  numbers  of  such  bacteria  which  actually  reach 
natural  waters  from  such  sources  are  seldom  large. 
The  count  at  37°,  therefore,  helps  to  distinguish  con- 
tamination by  wash  of  the  soil  of  a  virgin  woodland 
from  pollution  by  excreta,  since  in  the  former  case  the 
proportion  of  blood-temperature  organisms  is  much 
smaller  than  in  the  latter.  Furthermore,  this  method 
is  free  from  much  of  the  error  introduced  by  the  mul- 
tiplication of  bacteria  after  the  collection  of  a  sample, 
as  most  of  the  forms  which  grow  in  water  during  storage 
cannot  endure  the  higher  temperature  and  conse- 
quently do  not  develop  upon  incubation.  Recently, 
for  example,  water  from  a  spring  of  good  quality  was 
shipped  to  the  laboratory  from  a  considerable  distance. 
Gelatin  plates  showed  4200  bacteria  per  c.c.,  but  agar 
plates  at  37°  were  sterile. 

Significance   of   the   37°   Count.     A   majority  of   the 
English  Committee  appointed  to  consider  the  standard- 


DETERMINATION  OF  ORGANISMS  63 

ization  of  methods  for  water  examination  (1904) 
recommended  the  body-temperature  count  as  a  stand- 
ard procedure.  The  American  Committee  on  Standard 
Methods  in  its  1905  Report  did  not  recommend  this 
method  even  for  alternative  use.  In  its  last  report 
(1912),  however,  it  substituted  the  37°  for  the  20°  count, 
which  was  dropped  out  entirely.  As  we  have  pointed 
out  in  Chapter  II,  this  course  seems  to  us  an  unwise 
one,  and  it  was  formally  condemned  at  the  meeting  of 
the  Laboratory  Section  of  the  American  Public  Health 
Association  in  September,  1912,  by  the  passage  of  a  vote 
declaring  that  "  ordinary  routine  examinations  of 
water  for  sanitary  purposes,  and  in  the  control  of 
purification  plants  for  the  present,  should  include  the 
determination  of  the  number  of  bacteria  developing 
at  20  degrees  and  37  degrees."  By  this  action  the 
body-temperature  count  is  placed  on  a  par  with  the 
2o-degree  count  as  an  integral  part  of  sanitary  bac- 
teriological water  examination,  a  course  which  has 
been  strongly  urged  in  earlier  editions  of  this  book. 

The  body-temperature  count  must,  of  course,  be  made 
upon  agar  plates;  but  otherwise  the  procedure  is 
much  the  same  as  that  already  described  for  the  routine 
quantitative  bacteriological  examination  in  Chapter 
II.  A  1.5  per  cent  agar  medium  has  generally  been 
used,  but  the  Standard  Methods  Committee  in  its 
recent  report  recommends  only  i  per  cent  of  agar. 
Whipple  (1913)  points  out  that  this  i  per  cent  agar 
often  gives  trouble  from  the  running  together  of  the 
colonies  on  the  weaker  medium.  On  the  other  hand, 
a  i  per  cent  agar  gives  higher  counts  than  1.5  per  cent 


64         ELEMENTS  OF  WATER  BACTERIOLOGY 

agar.  He  emphasizes  the  recommendation  of  Jackson 
that  the  agar  used  should  be  dried  at  105°  C.  for 
30  minutes,  as  commercial  agar  itself  contains  more  or 
less  water. 

The  period  of  incubation  ordinarily  adopted  for  body- 
temperature  counts  is  24  hours.  Lederer  and  Bach- 
mann  (1911)  find  that  with  sewage  effluents  a  48-hour 
period  at  37°  may  yield  counts  from  two  to  six 
times  as  high  as  those  obtained  in  24  hours;  it  is  ques- 
tionable, however,  whether  the  higher  counts  thus  given 
would  compensate  for  the  loss  of  time.  The  adoption 
of  a  24-hour  period  by  the  Standard  Methods  Com- 
mittee in  any  case  represents  an  almost  universal 
practice. 

In  using  agar  plates  at  37°  difficulty  is  some- 
times caused  by  the  spreading  of  colonies  of  certain 
organisms  over  the  surface  of  the  plate  in  the  water 
of  condensation  which  gathers;  this  may  be  avoided 
by  inverting  the  plates  after  the  agar  is  once  well  set, 
or  still  better  by  the  use  of  plates  provided  with  earthen- 
ware tops,  as  suggested  by  Hill.  The  porous  earthen- 
ware absorbs  the  water  which  condenses  on  it,  the 
surface  of  the  plate  remains  comparatively  dry,  and  the 
percentage  of  "  spread "  plates  is  reduced  from  30 
per  cent  to  i  per  cent  (Hill,  1904).  Special  pains  must 
be  taken,  however,  to  keep  the  atmosphere  in  the 
incubator  nearly  saturated  with  moisture  or  errors  will 
be  introduced  by  the  excessive  evaporation  of  the 
medium  used. 

Use  of  Litmus  Lactose  Agar.  Additional  evidence 
as  to  the  character  of  a  water  sample  may  be 


DETERMINATION  OF  ORGANISMS  65 

obtained  with  little  extra  trouble  by  adding  a  sugar 
and  some  sterile  litmus  to  trie  agar  medium  and 
observing  the  fermenting  powers  of  the  organisms 
present,  as  first  suggested  by  Wurtz  (Wurtz,  1892) 
for  the  separation  of  B.  coli  from  B.  typhi.  It  hap- 
pens that  the  most  abundant  intestinal  organisms, 
belonging  to  the  groups  of  the  colon  bacilli  and  the 
streptococci,  decompose  dextrose  and  lactose  with  the 
formation  of  a  large  excess  of  acid.  The  decomposi- 
tion of  the  latter  sugar,  on  the  other  hand,  is  almost 
entirely  wanting  among  the  commoner  saprophytic 
bacteria,  and  therefore  lactose  is  most  commonly  used 
in  making  sugar  agar,  i  per  cent  being  added  to  the 
medium  just  before  the  final  filtration  (between  steps 
15  and  1 6  in  the  standard  process  of  media-making 
given  on  p.  102).  In  pouring  the  plate  a  cubic  centi- 
meter of  sterile  litmus  solution  should  be  added. 
After  incubation  the  colonies  of  the  acid-forming 
organisms  will  be  clearly  picked  out  by  the  redden- 
ing of  the  adjacent  agar.  Only  those  colonies  which 
are  sharply  colored  should  be  considered  as  significant, 
since  certain  bacteria  of  the  hay-bacillus  group  pro- 
duce weak  acid  and  faint  coloring  of  the  litmus. 

When  polluted  waters  are  examined  in  this  manner 
the  number  of  organisms  developing  on  the  lactose- 
agar  plate  will  be  very  high,  almost  equalling  in  some 
cases  the  total  count  obtained  on  gelatin.  Chick 
(Chick,  1901),  using  a  lactose-agar  medium  with  the 
addition  of  one-thousandth  part  of  phenol,  found,  of 
colon  bacilli  alone,  6100  per  c.c.  in  the  Manchester 
ship  canal;  55-190  in  the  polluted  River  Severn,  and 


66 


ELEMENTS  OF  WATEE  BACTERIOLOGY 


numbers  up  to  65,000  per  gram  in  roadside  mud.  In 
an  examination  of  water  from  the  Charles  River  above 
Boston,  37°  counts  ranging  from  9800  to  16,900  have 
been  found.  The  average  result  of  56  examinations  of 
Boston  sewage  from  July  to  December,  1903,  showed 
5,430,000  bacteria  per  c.c.,  at  20°  and  3,760,000  per 
c.c.  at  37°,  of  which  1,670,000  were  acid  formers.  The 
average  of  25  samples  examined  in  July  and  August, 
1904,  showed  1,690,000  bacteria  per  c.c.  at  20°  and 
1,400,000  at  37°;  429,000  per  c.c.  were  acid  formers 
(Winslow,  1905). 

In  unpolluted  waters  not  only  the  absolute  number  of 
organisms  developing  at  the  body  temperature  is  less,  but 
its  ratio  to  the  gelatin  count  is  very  different.  Rideal 
(Rideal,  1902)  states  that  the  proportion  between  the 
two  counts  in  the  case  of  a  London  water  in  a  year's 
examination  was  on  the  average  one  to  twelve.  Mathews 
(Mathews,  1893)  in  1893  gave  the  following  figures, 
the  contrast  between  the  ponds  and  streams,  which 
were  presumably  exposed  to  pollution,  on  the  one 
hand,  and  the  wells,  springs,  and  taps,  on  the  other, 
being  marked. 


Source  of  Water. 

Average  Number  of  Colonies  per  c.c. 

Gelatin,  20°. 

Wurtz  Agar,  37.5°. 

Wells  springs    

1664 

153 
296 
242 
273 

28 
43 
95 
24 

IOI 

Reservoirs 

Ponds 

Taps 

Streams  

DETEEMINATION  OF  ORGANISMS  67 

According  to  the  English  Committee  appointed  to 
consider  the  Standardization  of  Methods  for  the  Bac- 
terioscopic  Examination  of  Water  (1904),  the  ratio 
of  the  20°  count  to  the  37°  count  in  good  waters  is 
generally  considerably  higher  than  10  to  i.  "  With  a 
polluted  water  this  ratio  is  approached,  and  frequently 
becomes  10  to  2,  10  to  3  or  even  less." 

In  1903  Nibecker  and  one  of  ourselves  (Winslow 
and  Nibecker,  1903)  made  an  examination  of  259  samples 
of  water  from  presumably  unpolluted  sources  in  Eastern 
Massachusetts,  including  public  supplies,  brooks,  springs, 
ponds,  driven  wells,  and  pools  in  the  fields  and  woods, 
with  a  view  to  testing  the  value  of  the  body-temperature 
examination.  In  many  cases  the  samples  showed 
high  gelatin  counts,  since  some  of  the  waters  were 
exposed  to  surface  wash  from  vacant  land,  but  the 
average  number  of  organisms  developing  on  lactose 
agar  at  37  degrees  was  less  than  8  per  c.c.,  as  will  be 
seen  by  reference  to  the  table  on  the  following  page. 
The  highest  individual  counts  obtained  were  95  in  a 
meadow  pool,  83  in  a  brook,  and  74  in  a  barnyard  well, 
the  latter  probably  actually  polluted.  Only  two  samples 
in  the  whole  series,  one  from  the  well  above  mentioned, 
gave  any  red  colonies  on  the  agar  plates. 

For  a  series  of  shallow  surface  wells  recently  examined 
by  one  of  us  (S.  C.  P.)  a  similar  relation  is  indicated 
in  the  table  on  page  69;  124  samples  which  showed  no 
colon  bacilli  and  were  apparently  unpolluted,  gave  an 
average  of  190  bacteria  per  c.c.  at  20°  and  8  at  37° 
with  less  than  one  red  colony  per  c.c.;  23  samples 
which  did  contain  colon  bacilli  averaged  570  bacteria 


68 


ELEMENTS  OF  WATER  BACTERIOLOGY 


RELATION    OF    20°    AND    37°    COUNTS     IN    SAMPLES    OF 
WATER   FROM    APPARENTLY   UNPOLLUTED    SOURCES 

(WlNSLOW  AND  NlBECKER,   1903) 


Source  of  Samples. 

Number  of  Samples. 

"3  <8 
GJ3 

Litmus- 
lactose- 
agar 
Plates, 

37°. 

Dextrose-Broth 

Tubes. 

Average  Number 
of  Colonies. 

Average  Number 
of  Colonies. 

Plates  Showing 
Red  Colonies. 

<u 
*§ 

o 

15 

21 

18 

3 
18 

9 
18 
18 

15 

12 

9 
15 
6 

3 

45 
95 

45 

66 
65 

3 
6 
18 
3 
9 

21 

3 

*£ 
,0 

ii§ 

H  ° 
'o  rt 

0 

0 
0 

o 

0 
2 
0 

o 

0 
0 

o 

0 
0 
0 

0 

13 

I 

0 

o 

2 
0 
0 
0 
0 

5 

0 
0 

Number  of  Tubes 
with  Gas  2-1. 

Cambridge  supply  (tap)  
Wakefield  and  Stoneham  supply 
(tao) 

7 
6 
i 

6 

3 
6 

6 

5 
4 
3 
5 

i 
61 

11 

15 
i 

22 
TO 
I 

2 

6 

i 
3 

7 

i 

94 

59 
1  6 

35 
141 

36 
232 
13 

524 
4700 

223 
18 
294 
167 

365 
181 
811 

47 
188 

1235 
269 

15 

II 

6 

3 
18 

21 
I 

9 

M 

46 
8 

0 
12 

7 

i 

2 

9 
4 
3i 

4 
o 

0 

2 
27 

6 

2 
I 

O 
0 

2 
O 
0 
I    0 
0 
0 

o 
o 

0 
0 

o 

0 

o 
o 

0 

o 

0 
0 
0 
0 
0 
0 
0 

0 
0 

0 
0 

o 
o 

0 
0 
0 
0 

3 

0 
0 

o 

13 

2 
0 

0 
0 

o 
5 

0 

0 

0 
0 
0 
0 
0 
0 
0 
0 
0 

3 

0 
0 
0 
0 
0 

o 

0 
0 
0 

o 
o 

0 

o 

0 

o 

0 

o 

0 

Lynn  supply  (tap)  

Brookline  supply  (tap) 

Plymouth  supply  (tap)  
Peabody  supply  (tap)  
Dedham  supply  (tap)  
Newburyport  supply  (tap)  
Salem  supply  (tap)  
Taunton  supply  (tap)  
Sharon  (well)  (tap)  
Medford  supply  (tap)  
Milton  supply  (tap)  
Westerly,  R.  I.,  supply  (tap)  
Brooks  

Driven  wells  
Springs  
Ponds  fed  by  brooks  
Melted  snow 

Pools  in  fields  
Pools  in  woods  

Roadside  pool^ 

Stream.  Blue  Hill  Reservation.  .  .  . 
Flow  from  rocks  
Ponds  fed  by  springs  
Drainage  from  manured  pasture  .  . 
Swamps  
Rain-water   after    twelve    hours' 
heavy  fall  
Shallow  well  in  Lynn  woods  

Totals  

2  SO 

.        ..    J4 

775 

41 

38 

3 

DETERMINATION  OF  ORGANISMS 


69 


per  c.c.  at  20°  and  55  at  37°  with  an  average  of  7  red 
colonies. 


BACTERIAL  CONTENT  OF   147  SHALLOW  WELLS 

PERCENTAGE  OF  SAMPLES  IN  EACH  GROUP 

Bacteria  per  c.c. 


B. 

coli. 

o 

I-IO 

11-20 

21-50 

51- 

100 

101- 
500 

501- 

IOOO 

1001- 

2OOO 

2001- 
3OOO 

_ 

Gelatin,  20° 

3 

16 

14 

16 

II 

31 

5 

4 

+ 

5 

10 

57 

10 

14 

5 

— 

Agar,  37° 

IS 

63 

10 

10 

I 

i 

+ 

3i 

35 

22 

4 

4 

4 

— 

Red  colonies 

86 

12 

2 

+ 

3_o 

52 

9 

9 

Significance  of  High  Temperature  Counts.  Important 
data  as  to  the  distribution  of  bacteria  which  will  develop 
at  high  temperatures  may  be  found  in  a  paper  by 
Gage  (1906),  coupled  with  a  suggestive  discussion  of  the 
general  significance  of  bacterial  ratios.  The  table  on  page 
70  shows  some  of  the  most  significant  results  obtained  by 
plating  waters  of  various  degrees  of  purity  at  20°,  40°  and 
50°.  We  have  rearranged  the  lines  of  the  table  so  as  to 
make  the  progression  from  more  to  less  polluted  waters 
a  fairly  regular  one.  The  colony  count  at  50°  shows  an 
even  sharper  differentiation  than  that  at  40°.  Gage 
rightly  concludes  that  "  the  information  to  be  obtained 
by  counts  of  bacteria  and  acid-producing  organisms  at 
any  one  of  the  above  temperatures  is  greatly  increased 
by  the  combination  of  the  results  obtained  from  counts 
at  two  or  more  temperatures." 


70 


ELEMENTS  OF  WATER  BACTERIOLOGY 


AVERAGE  NUMBER  OF  BACTERIA  AND  ACID-PRODUCERS 
DEVELOPING  AT  20°,  40°,  AND  50°  C.,  WITH  DIFFER- 
ENT CLASSES  OF  WATERS 


Bacteria  per  c.c. 

Acid-producing  Bacteria. 

20°  C. 

4D. 

40°  C. 
24  Hrs. 

50°  C. 

24  Hrs. 

20°   C. 

4D. 

40°  C. 
24  Hrs. 

50°  C. 

24  Hrs. 

Sewage 

2,990,000 
1,676,000 
485,000 
146,600 
389,000 
306,000 

15,500 
23,300 
16,400 
16,900 
2,800 

1,640 

35 
1,300 
670 

32 
7i5 
62 

150 
64 
i  ,000 

507 

27 

7i 
49 
80 

4i 

557,500 
360,000 
126,500 
26,IOO 
59,300 
89,600 

1,730 
2,030 
112 

207 
212 

i,375 
4 
130 
170 

3 
170 

i 

22 

5 

72 

I 

8 
o 

0 

7,700 
29,500 
410 
8,300 
8,000 
485 

154 
54 
5 
4 

2 

2 
0 

I 
2 

I 

0 
I 

I 

o 

0 

o 

0 
0 

o 

0 

1,940,000 
1,032,000 
241,000 
112,400 
292,000 
193,000 

15,200 
I6.OOO 
6,700 
2,500 
1,650 

2,360 
29 
345 
1,045 

6 

259 
16 

14 
ii 

82 
8 
30 
6 
8 

0 

346,000 
283,000 
90,000 
22,700 
45,000 
46,000 

1,360 
1,180 
87 
134 
66 

i,i95 

2 
119 
154 

I 
IOI 
O 

17 

3 

i 

55 
i 

r 
O 
2 
0 

4,400 
24,900 
240 
8,000 
8,000 
200 

100 

20 

2 
2 

I 

I 
O 
O 

o 

I 
I 
o 

I 
I 

0 

o 
o 

0 

o 
o 

0 

Septic  effluent  
Contact  effluent  .  . 

«             (i 

Trickling       filter 
effluent  
Do  

Canal  water  
River  water  
Settled  canal  water 
Sand  filter  effluent 
(sewage)  
Do  

Do  

Do 

Water  filter  efflu- 
ent   
Do 

Do  
Do  

Do 

Shallow  well  

(  (               a. 

Pond 

<  i 

Spring  

i  ( 

Driven  well  

In  warm  weather  the  interpretation  of  the  body- 
temperature  count  must  be  made  less  rigid  than  at 
other  seasons.  Recent  investigations  have  shown  that 


DETERMINATION  OF  ORGANISMS 


71 


in  midsummer  bacteria  capable  of  growth  at  37°  are 
more  abundant  in  normal  waters  than  in  winter  and 
spring. 

Winslow  and  Phelps  examined  86  samples  from 
springs,  wells,  brooks  and  pools  during  the  winter 
and  spring  months  and  found  only  12  which  showed 
more  than  25  bacteria  per  c.c.  and  only  3  which  showed 
more  than  100  per  c.c.  on  lactose-agar.  On  the  other 
hand,  of  58  samples  from  corresponding  sources 
examined  in  summer,  16  contained  more  than  100 
bacteria  per  c.c.  A  series  of  20  pools,  ponds,  and  brooks 
at  Mt.  Desert,  Me.,  which  were  entirely  free  from 
human  or  animal  pollution,  were  examined  in  the  late 
summer  of  1906.  Only  4  of  the  20  samples  gave 

20°  AND  37°  COUNTS  OF  RAW  WATER   AT  WILMINGTON 
FILTER  PLANT 

(WHIPPLE,  1913) 


1908. 

1909. 

Month. 

Bacteria  per  c.c. 

Per 
Cent 

Bacteria  per  c.c. 

Per 
Cent. 

Gelatin, 

20°. 

Bile-agar, 
37°. 

Gelatin, 

20°. 

Bile-agar, 
37°. 

January      

4630 
6830 
8800 
3170 
2OIO 
1640 
3150 
3140 
3400 
5180 
6850 
4IOO 

124 
358 
350 
149 
119 
241 
432 
451 
644 

439 

78 
203 

2.7 

5-3 
4.0 

4-7 
5-9 
14-7 
13-7 
14.4 
18.9 
8-5 

I  .  2 

4-9 

3880 
4800 
4620 
5080 
3340 
2350 
2940 
1430 
2620 
1380 
1650 
4150 

94 
260 

387 
347 
229 
158 
57 
230 
619 
129 

97 
194 

2-3 
5-4 
8-4 
6-7 
6.8 

6-7 
1.9 
16.  i 

22.8 

9-4 
3-9 

4-7 

February 

March  

April 

May      

Tune 

July 

August  

September  

October  

November. 

December  

72 


ELEMENTS  OF  WATER  BACTERIOLOGY 


counts  under  25  at  37°,  and  7  of  them  gave  counts 
over  100,  the  highest  figure  being  425. 

Whipple  (1913)  gives  some  figures  for  the  raw  water 
at  the  Wilmington,  Del.,  filter  plant  (page  71)  which 
bring  out  the  seasonal  variation  very  clearly. 

Another  special  case  in  which  the  ratio  between  the 
20°  and  the  37°  count  fails  to  be  significant  is  that 
of  a  water  which  has  been  treated  with  bleaching 
powder.  Most  of  the  bacteria  which  survive  chlorine 
treatment  are  of  course  spore  formers,  many  of  them 
belonging  to  the  hay  bacillus  group,  and  it  happens 
that  most  of  these  spore  formers  can  grow  at  body 
temperature.  Thus  it  is  common  to  get  counts  as  high 
at  37°  as  at  20°  with  such  waters,  although  the  absolute 
numbers  are  generally  small.  This  point  is  illustrated 
in  the  two  tables  below,  showing  the  results  of  experi- 
mental treatment  of  Merrimac  River  water  at  the 
Lawrence  Experiment  Station  and  of  swimming  pool 
water  at  the  University  of  Wisconsin. 


COMPARATIVE  EFFECTS  OF  CHLORINE  DISINFECTION 
UPON  20°  AND  37°  COUNTS,  MERRIMAC  RIVER 
WATER,  AT  LAWRENCE,  MASS. 

(CLARK  AND  GAGE,  1909) 


Bacteria  per  c.c. 

, 

Untreated  Water. 

Treated  Water. 

Sample. 

20°. 

37°. 

20°. 

37°. 

A 

3,400 

30 

12 

4 

B 

28,900 

130 

4 

4 

C 

14,000 

75 

35 

47 

D 

3,700 

81 

43 

62 

DETERMINATION  OF  ORGANISMS 


73 


COMPARATIVE  EFFECTS  OF  CHLORINE  DISINFECTION 
UPON  20°  AND  37°  COUNTS,  SWIMMING  POOL  WATER 
AT  UNIVERSITY  OF  WISCONSIN 

(TULLY,  1912) 


Bacteria  per  c.c. 

Untreated  Water. 

Treated  Water. 

Sample. 

20°. 

37° 

20°. 

37°. 

A 

275 

16 

0 

I 

B 

445 

480 

4 

5 

C 

920 

483 

8 

8 

D 

5)630 

680 

4 

2 

E 

19,100 

1,140 

30 

45 

F 

24,000 

1,190 

130 

120 

G 

IO,OOO 

1,  080 

14 

27 

H 

1,700 

690 

15 

9 

I 

2,570 

78o 

12 

30 

J 

2,800 

560 

27 

66 

Under  ordinary  conditions  it  is  clear  that  organisms 
growing  at  the  body  temperature  and  those  fermenting 
lactose  are  not  numerous  in  normal  waters.  The 
absolute  count  at  37°  seldom  exceeds  50,  and  is  rarely 
over  10  per  cent  of  the  20°  count,  except  after  hot 
periods  in  the  late  summer ;  acid  producers  are  generally 
entirely  absent.  On  the  other  hand,  the  numbers  on 
the  litmus-lactose-agar  plate  will  be  likely  to  run  into 
hundreds  with  a  good  proportion  of  red  colonies  when 
polluted  waters  are  examined. 


CHAPTER  V 

THE  ISOLATION  OF  SPECIFIC  PATHOGENES  FROM 
WATER 

THE  discovery  of  the  organisms  which  specifically 
cause  infectious  diseases  naturally  led  to  the  hope 
that  their  isolation  from  polluted  water  might  become 
the  most  convincing  proof  of  its  sanitary  quality.  The 
typhoid  bacillus  and  the  spirillum  of  Asiatic  cholera 
were  in  this  connection  of  paramount  importance,  and 
to  the  search  for  them  many  investigators  have  devoted 
themselves. 

The  Search  for  Typhoid  Bacilli.  In  the  earlier  exam- 
inations of  water  for  the  typhoid  bacillus  an  attempt 
was  made  to  use  media  which  especially  favored  the 
growth  of  the  microbe  sought  for,  or  to  begin  with 
some  process  of  "  enrichment  "  in  which  the  sample 
was  incubated  under  conditions  which  would  favor 
the  growth  of  the  pathogenic  organisms  while  check- 
ing the  development  of  the  common  water  bacteria. 
It  was  apparent  that  the  body  temperature  and  the 
presence  of  a  slight  excess  of  free  acid  furnished  such 
conditions,  and  most  of  the  methods  suggested  rest 
upon  these  principles.  Among  them,  one  of  the  earliest 
was  that  of  Parietti  (Parietti,  1890),  which  consists 
in  the  addition  of  the  water  to  a  series  of  broth  tubes 

74 


ISOLATION  OF  SPECIFIC  PATHOGENES         75 

containing  increasing  amounts  of  a  solution  of  4  per 
cent  hydrochloric  acid  and  5  per  cent  phenol.  From 
tubes  in  which  growth  occurs  after  24  hours  at  37  degrees 
the  organisms  present  may  be  isolated  in  pure  cultures 
by  some  plating  method  and  identified  by  subcultures. 

The  great  difficulty  with  a  majority  of  the  enrichment 
processes  is  that  the  conditions  which  favor  the  multipli- 
cation of  the  typhoid  bacillus  are  frequently  suited  in  an 
even  higher  degree  to  B.  coli  and  other  intestinal  organ- 
isms. Being  present  in  almost  all  cases  in  much 
higher  numbers  than  B.  typhi,  these  bacteria  develop 
more  abundantly,  and  effectually  mask  any  disease 
germs  originally  present.  In  order  to  obviate  this 
difficulty,  Hankin  (Hankin,  1899),  after  adding  suc- 
cessively increasing  portions  of  Parietti  solution  to 
tubes  inoculated  with  the  water  to  be  tested,  selected 
the  second  highest  tube  of  the  series  in  which  growth 
occurred  for  the  inoculation  of  a  new  set,  finally  plating 
as  above.  He  believed  that  the  chance  for  overgrowth 
by  this  method  is  somewhat  decreased;  but  in  the  hands 
of  other  investigators  it  has  not  met  with  marked  suc- 
cess. Klein  (Thomson,  1894)  in  his  investigations, 
made  use  of  the  Berkefeld  filter  to  concentrate  the 
organisms  in  the  sample.  Some  observers  abandoned 
the  enrichment  process  altogether  and  recommended 
direct  plating  upon  solid  media  such  as  phenolated 
gelatin  or  the  Eisner  (Eisner,  1896)  medium,  made  by 
adding  10  per  cent  of  gelatin  and  i  per  cent  of  potas- 
sium iodide  to  an  infusion  of  potato  whose  reaction 
has  been  adjusted  to  30  on  Fuller's  scale. 

In  the  last  five  years  considerable  progress  has  been 


76          ELEMENTS  OF  WATER  BACTERIOLOGY 

made  in  the  development  of  new  methods  for  isolating 
the  typhoid  bacillus.  These  fall  in  three  distinct 
groups:  first,  the  direct  isolation  by  differential,  fre- 
quently colored,  solid  media;  second,  isolation  as 
above,  preceded  by  enrichment  methods;  third,  isola- 
tion, with  or  without  enrichment,  preceded  by  concen- 
tration of  the  organisms  by  agglutination  with  typhoid 
serum  or  concentration  by  chemical  precipitation. 

Isolation  Methods,  Using  Solid  Media.  Drigalski 
and  Conradi  (Drigalski  and  Conradi,  1902),  prepared 
a  medium  primarily  for  the  isolation  of  typhoid  bacilli 
from  excreta,  which  may  also  be  applied  in  water 
bacteriology.  This  consists  of  an  agar  medium 
containing  nutrose,  sodium  chloride,  litmus,  lactose, 
and  a  dye,  "  crystal  violet ";  and  it  is  used  in  the  form 
of  plate  cultures  infected  by  smearing  the  surface  with 
a  bent  glass  rod  after  thorough  cooling.  The  culture 
medium  is  a  selective  one,  ordinary  saprophytes  fail- 
ing to  grow,  while  after  14  to  24  hours  at  37°, 
colon  and  typhoid  colonies  can  be  readily  distinguished 
from  one  another.  The  colon  bacillus  produces  red, 
non-transparent  colonies,  of  variable  size  and  depth 
of  color,  while  the  typhoid  colonies  are  blue  or  violet, 
transparent  and  of  smaller  size,  seldom  exceeding  three 
millimeters  in  diameter. 

Endo  (Endo,  1904)  has  suggested  the  use  of  a  fuchsin- 
lactose-agar  decolorized  by  sodium  sulphite.  Upon 
this  medium  B.  coli  produces  bright  red,  sharply  defined 
round  colonies  in  24  hours  at  37°,  while  B.  typhi 
gives  round,  colorless,  transparent  colonies  with  thin 
margins.  This  medium  has  been  somewhat  modified 


ISOLATION  OF  SPECIFIC  PATHOGENES         77 

by  Gaehtgens  (Gaehtgens,  1905)  by  the  addition  of 
caffein,  and  he  found  it  of  great  service  in  isolating  the 
typhoid  bacillus  from  stools  of  patients  suffering  with 
the  disease.  No  attempts  were  made  by  him  to  isolate 
the  organism  from  polluted  water. 

Loeffler  (Loeffler,  1903  and  1906)  and  Lentz  and 
Tietz  (Lentz  and  Tietz,  1903  and  1905)  have  made  use 
of  an  agar  medium  containing  malachite  green.  This 
medium  is  supposed  to  inhibit  the  growth  of  B.  coli 
while  favoring  B.  typhi,  and  has  been  recommended 
for  the  isolation  of  the  organism  from  faeces.  Dcebert 
(Dcebert,  1900)  has  shown  that  certain  varieties  of 
ma!achite  green  are  not  suited  to  this  purpose.  Nowack 
(Nowack,  1905)  has  also  pointed  out  the  same  fact, 
and  ascribed  the  difference  to  the  presence  of  dextrin. 
He  also  showed  that  a  medium  0.8  per  cent  alkaline  to 
phenol-phthalein  is  more  favorable  to  B.  typhi  and 
less  favorable  to  B.  coli  than  one  neutral  to  litmus. 
With  such  a  medium  he  found  that  about  20  per  cent 
of  the  typhoid  bacilli  present  develop.  Lemke  (1911) 
has  recently  reported  good  success  in  isolating  typhoid 
and  para-typhoid  bacilli  from  artificially  infected 
waters  by  the  use  of  nutrient  broth  containing  3-5 
per  cent  of  sodium  chloride  and  varying  amounts  of 
malachite  green  as  an  enrichment  medium. 

The  use  of  the  inhibitive  anilin  dyes  like  crystal 
violet  and  malachite  green  has  the  disadvantage  of  also 
inhibiting  to  some  extent  the  development  of  the  weaker 
typhoid  organisms.  Another  principle  is  involved  in 
the  media  proposed  by  Hiss  and  Hesse.  These  are 
both  agar  media  of  lower  spissitude  than  ordinary 


78         ELEMENTS  OF  WATER  BACTERIOLOGY 

agar  and  the  separation  of  the  typhoid  and  colon  groups 
of  organisms  depends  on  the  greater  motility  of  the 
former  and  their  tendency  to  swim  out  from  the  colonies 
and  form  branch-like  processes  or  turbid  zones  on  a 
semi-solid  medium.  The  Hiss  medium  is  made  up  as 
follows  (Hiss,  1902): 

Agar 15  gm.     NaCl 5  gm. 

Gelatin 15  gm.     Dextrose 10  gm. 

Liebig's  meat  extract    5  gm.     Distilled  water  1000  c.c. 
Reaction  i  per  cent  normal. 

The  Hesse  medium  has  been  used  with  great  success 
by  Jackson  and  Melia  (1909).  Its  general  composition 
is  as  follows: 

Agar 5  gm.     NaCl 8.5  gm. 

Witte's  pepton 10  gm.     Distilled  water  1000  c.c. 

Liebig's  meat  extract    5  gm. 
Reaction  i  per  cent  normal. 

Jackson  (1909)  recommends  that  the  agar  used 
should  be  dried  for  half  an  hour  at  105°,  and  under 
these  circumstances  4.5  grams  may  be  used  in  the 
formula  instead  of  the  5  grams  recommended  by  Hesse. 
The  medium  must  be  stored  in  an  ice  chest  with  saturated 
atmosphere.  Plates  must  be  made  in  sufficient  dilu- 
tion to  give  a  few  colonies  on  the  plate;  and  where 
this  is  done  the  typhoid  colonies  are  sharply  dis- 
tinguished from  those  of  B.  coli  by  the  fact  that  they 
grow  to  a  considerable  size,  often  several  centimeters 
in  diameter  and  show  a  broad  translucent  or  scarcely 


ISOLATION  OF  SPECIFIC  PATHOGENES         79 

turbid  zone  between  the  white  opaque  centre  or  nucleus 
and  the  perfectly  circular  narrow  white  seam  or  edge. 

Stokes  and  Hachtel  (1912)  have  suggested  a  modifica- 
tion of  the  Hesse  medium  which  consists  in  the  increase 
of  the  agar  to  5.5  per  cent  and  in  the  addition  of  10 
gm.  of  lactose  and  50  gm.  of  glycerin  to  the  formula 
cited  above.  The  agar  is  dried  out  at  105  degrees  for 
half  an  hour  and  dissolved  in  half  a  liter  of  water.  The 
meat  extract  is  added  to  the  other  half  liter  and  freed 
from  muscle  sugar  by  inoculation  with  the  colon  bacillus 
and  incubation  for  24  hours  at  37  degrees.  The  sugar- 
free  broth  thus  prepared  is  filtered  and  to  it  is  added 
the  peptone,  lactose  and  salt.  The  two  half  liter  por- 
tions of  the  medium  are  then  mixed  and  boiled  for 
30  minutes.  The  medium  is  filtered  and  adjusted  to  a 
neutral  reaction,  the  glycerin  is  added  and  the  medium 
is  tinted  with  azolitmin  solution  before  tubing  and  steril- 
izing. Typhoid  and  para-typhoid  organisms  develop 
medium-sized,  pinkish  colonies  with  concentric  rings, 
and  may  thus  be  distinguished  from  colonies  of  B. 
alcaligenes,  B.  proteus  and  other  motile  forms.  Organ- 
isms of  the  B.  subtilis  group  must  be  eliminated  by 
microscopic  examination,  using  the  Gram  stain. 

Preliminary  Enrichment.  In  most  cases  plate  isola- 
tion is  preceded  by  some  sort  of  enrichment  process 
designed  to  favor  the  typhoid  bacilli  at  the  expense 
of  the  members  of  the  B.  coli  group.  The  original 
use  of  carbol  broth  has  been  already  discussed.  In 
Europe  caffein  media  have  been  used  for  this  purpose 
and  in  the  United  Staes  bile  media  have  been  strongly 
recommended. 


80         ELEMENTS  OF  WATER  BACTERIOLOGY 

The  important  fact  that  caffein  has  an  inhibitory 
action  on  colon  bacilli,  announced  by  Roth  (Roth, 
1903)  has  given  rise  to  much  investigation,  and  offers 
one  of  the  most  promising  methods  for  the  isolation 
of  the  typhoid  bacillus  from  water.  Hoffman  and 
Ficker  (Hoffman  and  Ficker,  1904)  developed  methods 
for  the  isolation  of  B .  typhi  from  faeces  and  from  infected 
water  by  its  use  in  connection  with  nutrose  and  crystal 
violet.  For  the  isolation  from  infected  water  solutions 
were  prepared  as  follows : 

1.  Ten  grams  of  nutrose  dissolved  in  80  c.c.  of  steril- 
ized distilled  water. 

2.  Five  grams  caffein,  in    20  c.c.  sterilized  distilled 
water. 

3.  One-tenth  gram  of  crystal  violet  in  100  c.c.  water. 
Solutions  i  and  2  were  mixed  by  shaking  together  in  a 
flask,  and  the  mixture  poured  into  a  flask  containing 
900  cubic  centimeters  of  the  water  to  be  tested;    10 
c.c.  of  solution  3  were  gradually  added,  and  the  whole 
thoroughly  mixed  by  shaking  and  then  incubated  at 
37  degrees  for  not  over  12-13  hours.     At  the  end  of  the 
incubation  period  loopfuls  of  the  solution  were  smeared 
over  Drigalski-Conradi  plates. 

By  this  method  the  B.  typhi  was  isolated  from 
mixtures  in  river  water  containing  one  typhoid  bacillus 
to  51,867  water  bacteria  and  colon  bacilli. 

A  number  of  investigations  have  shown  that  the 
action  of  the  caffein  is  not  as  markedly  selective  as  at 
first  claimed.  Kloumann  (Kloumann,  1904)  obtained 
no  better  results  by  this  method  than  by  the  Drigalski- 
Conradi  medium  alone,  and  Willson  (Willson,  1905) 


ISOLATION  OF  SPECIFIC  PATHOGENES         81 

found  that  certain  strains  of  B.  typhi  were  inhibited, 
while  strains  of  B.  coli  developed  feebly  in  the  presence 
of  0.5  per  cent  of  caffein. 

In  this  country  marked  success  in  the  isolation  of 
the  typhoid  bacillus  has  been  attained  by  the  use  of 
lactose  bile  as  an  enrichment  medium.  Jackson  (Jack- 
son and  Melia,  1909),  the  principal  exponent  of  this 
procedure,  recommends  that  sterilized  undiluted  fresh 
ox  gall  (or  an  ii  per  cent  solution  of  dry  fresh  ox  gall) 
containing  i  per  cent  of  peptone  and  i  per  cent  of 
lactose  be  made  up  in  40  c.c.  amounts  in  fermen- 
tation tubes  to  which  varying  amounts  of  water,  up 
to  10  c.c.  may  be  added.  After  incubation  for  48 
or  72  hours,  he  plates  on  Hesse  a  gar  and  he  finds 
that  in  the  bile  medium  B.  typhi  tends  to  over- 
grow B.  coli  while  most  other  organisms  are  entirely 
suppressed. 

Concentration  by  Agglutination  or  Precipitation.  A 
physical  concentration  of  the  typhoid  bacilli  precedes 
enrichment  or  isolation  in  the  procedure  recommended 
by  many  authors.  Klein,  as  noted  above,  accomplished 
this  by  passing  the  water  through  a  Berkefeld  filter. 
Other  workers  have  made  use  of  agglutination  or  chem- 
ical precipitation  for  the  same  purpose. 

The  phenomenon  of  agglutination  was  made  the 
basis  of  a  method  of  isolating  B.  typhi  from  water  by 
Adami  and  Chopin  (Adami  and  Chopin,  1904).  Two- 
liter  samples  of  the  water  were  collected  in  sterilized 
bottles  (Winchester  quarts),  and  to  each  was  added 
20  c.c.  of  i  per  cent  glucose  broth.  The  sample  was 
incubated  for  18  to  24  hours  at  37°  C.,  after  which 


82         ELEMENTS  OF  WATER  BACTERIOLOGY 

10  c.c.  portions  were  withdrawn  and  placed  in  long, 
narrow  test  tubes.  To  each  of  these  tubes  enough 
typhoid  serum  of  known  potency  was  added  to  make 
a  regularly  graded  series,  1-50,  i-ioo,  1-150,  and  1-200. 
The  probable  presence  of  the  typhoid  bacillus  was 
manifest  by  the  formation  of  flocculi  within  a  quarter 
of  an  hour,  and  agglutination  was  complete  in  from 
2  to  5  hours. 

The  tube  having  the  greatest  dilution  in  which 
agglutination  was  apparent  was  then  examined  by 
breaking  off  the  lower  end,  containing  the  precipitate, 
washing  the  sediment  two  or  three  times  with  sterile 
water  after  removing  the  clear  supernatant  liquid, 
and  allowing  the  bacteria  to  settle  again.  The  organ- 
isms remaining  were  plated  upon  various  media,  and 
examined  biochemically  to  determine  the  true  character 
of  the  suspected  colonies.  It  was  found  that  a  dilution 
of  i  to  60  was  the  highest  which  could  be  used  with  the 
organisms  examined,  and  it  is  therefore  probable  that 
high  dilutions  (greater  than  1-60)  cannot  be  success- 
fully used. 

Investigation  of  an  organism  isolated  by  this  method 
was  made  by  Klotz  (1904),  who  found  the  culture 
to  be  not  a  typical  B.  typhi,  but  a  form  showing 
certain  points  of  similarity  to  both  B.  typhi  and  to 
B.  coli,  and  probably  intermediate  between  them. 
Frost  (1910)  isolated  a  bacillus  of  the  B.  proteus 
group  from  filtered  Potomac  water  which  agglutinated 
with  typhoid  serum  in  high  dilutions.  As  Klotz  points 
out,  therefore,  it  is  evident  that  even  when  a  positive 
result  is  obtained  with  a  relatively  high  dilution  of 


ISOLATION  OF  SPECIFIC  PATHOGENES         83 

typhoid  serum,  the  action  may  by  no  means  be  abso- 
lutely specific. 

Schepilewski  (Schepilewski,  1903)  and  Altschuler 
(Altschuler,  1903)  have  also  used  agglutination  as  a 
means  of  precipitating  the  bacteria  after  enrichment 
cultivation  in  broth.  The  former  incubated  the  cul- 
ture at  37°  for  24  [hours,  then  added  a  serum  of 
high  potency,  allowed  the  mixture  to  stand  for  2 
to  3  hours,  and  then  centrifuged.  The  supernatant 
liquid  was  removed,  and  the  mass  of  agglutinated  cells 
broken  up  by  shaking  with  glass  beads  and  salt  solu- 
tion. Upon  plating  upon  litmus  lactose  agar  the  organ- 
isms could  be  detected.  In  this  way  positive  isolation 
was  made  from  water  containing  i  loopful  of  a  broth 
culture  in  50  liters  of  water.  Altschuler's  method  of 
enrichment  was  essentially  like  that  of  Schepilewski. 
From  the  surface  of  the  culture  developed  at  37°, 
10  c.c.  were  removed  to  a  tapering  tube  provided  with  a 
rubber  tube  at  the  bottom.  Serum  was  added  in  the 
proportion  of  one  part  in  50,  the  culture  agitated  to 
release  entangled  non-agglutinated  bacilli  and  the 
sediment  run  into  a  tube  containing  i  per  cent  peptone 
and  0.5  per  cent  salt.  The  agglutinated  mass  was  broken 
up  by  shaking  with  sand,  and  the  culture  incubated  at 
37°  for  24  hours,  then  plated  on  Drigalski-Conradi 
plates.  The  organism  was  isolated  from  dilute  suspen- 
sions in  water  (150  in  i  liter)  and  also  from  the  faeces 
of  a  typhoid  patient  from  which  other  methods  gave 
negative  results. 

Precipitation  Methods.      A  number  of  methods  for 
concentrating    typhoid   bacilli    in    water    by    chemical 


84         ELEMENTS  OF  WATER  BACTERIOLOGY 

precipitation  have  been  tested  experimentally,  with 
some  degree  of  promise.  Vallet  (Vallet,  1901)  was 
the  first  to  employ  this  principle,  and  made  use  of 
sodium  hyposulphite  and  lead  acetate.  The  mixture 
was  then  centrifuged  and  the  precipitate  dissolved  in 
more  hyposulphite.  The  clear  solution  was  then  plated. 

Schlider  (Schiider,  1903)  observed  that  the  lead 
salt  reacted  harmfully  upon  the  bacteria,  and  that 
the  hyposulphite  should  be  in  excess.  In  his  experi- 
ments water  was  allowed  to  stand  in  tall  jars  for  24 
hours.  To  2  liters  of  infected  water,  20  c.c.  of  a  7.75 
per  cent  solution  of  sodium  hyposulphite  was  added, 
and  after  thorough  mixing  20  c.c.  of  a  10  per  cent 
solution  of  lead  nitrate.  The  precipitate,  after  20 
to  24  hours,  was  treated  with  14  c.c.  of  saturated  sodium 
hyposulphite  solution  and  shaken.  From  the  clear 
solution  0.2  to  0.5  c.c.  portions  were  streaked  upon 
Drigalski-Conradi  plates  which  were  then  incubated 
at  37°  for  24  hours.  Ficker  (Ficker,  1904)  modi- 
fied the  process  still  more  by  using  ferric  sulphate, 
and  dissolved  the  precipitate  with  neutral  potassium 
tartrate.  The  final  solution  was  then  plated  on 
Drigalski-Conradi  medium.  Ficker  claimed  that  this 
method  gives  excellent  results,  97-98  per  cent  of  the 
typhoid  bacteria  being  carried  down  with  the  precipitate. 

Miiller  (Miiller,  1905),  in  comparing  different  pre- 
cipitation methods,  adopted  ferric  oxychloride  as  the 
most  suitable  precipitant,  because  of  its  quicker  and 
less  destructive  action.  Willson  (Willson,  1905)  sug- 
gested the  use  of  alum  as  a  precipitant.  He  added 
0.5  gr.  alum  per  liter  of  water  examined.  The  mixture 


ISOLATION  OF  SPECIFIC  PATHOGENES         85 

was  then  centrifuged,  and  the  precipitate  suspended  in  a 
small  amount  of  water  and  plated  on  Drigalski-Conradi 
medium.  Nieter  (Nieter,  1906)  made  20  parallel  experi- 
ments, using  very  pure  water  infected  with  typhoid 
bacilli  in  varying  numbers.  By  precipitating  with  ferric 
sulphate  and  sodium  hydrate,  centrifuging,  and  then 
filtering  through  a  sterile  filter  he  obtained  small 
numbers  of  bacteria.  Using  iron  oxychloride  as  the 
precipitant,  he  confirmed  the  results  of  Miiller.  By 
plating  on  malachite  green  agar  he  was  often  able  to 
get  positive  results  when  the  Drigalski-Conradi  medium 
failed. 

By  use  of  a  combination  of  enrichment  and  chemical 
precipitation,  Ditthorn  and  Gildemeister  (Ditthorn 
and  Gildemeister,  1906)  isolated  the  typhoid  bacillus 
from  enormous  artificial  dilutions  in  water.  In  the 
typhoid  fever  epidemic  in  Posen,  in  1906,  it  was  found 
that  the  bile  of  those  dying  from  the  disease  contained 
nearly  pure  cultures  of  typhoid  bacilli.  This  led  the 
authors  mentioned  to  use  bile  and  bile  agar  as  enrich- 
ment^media.  After  precipitating  by  Miiller's  method, 
the  whole  of  the  precipitate  was  added  to  100 
c.c.  sterile  ox  bile  and  grown  at  37°  for  24  hours, 
after  which  time  i  c.c.  portions  were  plated.  With 
extreme  dilutions  it  was  found  desirable  to  incubate 
for  48  to  72  hours.  The  results  were  unsatisfactory 
in  the  presence  of  large  numbers  of  water  bacteria. 
It  is  also  pointed  out  that  the  iron  oxychloride  is 
bactericidal  in  48  hours. 

Separation  on  the  Basis  of  Motility.  Drigalski  (Dri- 
galski,  1906)  has  suggested  the  separation  of  B.  typhi 


86 


ELEMENTS  OF  WATER  BACTERIOLOGY 


from  other  bacteria  in  water  through  its  greater  motility. 
He  succeeded  in  isolating  typhoid  bacilli  from  two 
springs  by  the  following  method:  5  to  10  liters  of  water 
were  allowed  to  stand  one  to  two  days  in  tall  milk  cans 
at  room  temperature.  Samples  were  taken  from  the 
surface  and  plated  on  litmus-lactose  agar  (Drigalski- 
Conradi  medium),  the  amount  of  water  to  be  used 
varying  with  the  contamination. 

Starkey  (1906)  has  suggested  the  use  of  an  apparatus 
consisting  of  a  piece  of  glass  tubing  bent  so  as  to  give 
four  successive  connected  loops.  This  is  filled  with  phenol 
broth,  inoculated  atone  end,  and  incubated  anaerobically. 
The  more  actively  motile  bacilli  find  their  way  to  the 
fourth  loop  from  which  they  may  be  isolated  by  plating. 

Review  of  Suggested  Procedures.  The  methods  of 
examining  water  for  B.  typhi  may  be  conveniently 
summarized  as  follows: 


a.  By  filtration 

b.  By  agglutination 

f  Schiider's  process 

c.  By  chemical  I  Fischer's  process 
precipitation  1  Willson's  process 

(  M  tiller's  process 
a.  Hoffmann  and  Ficker's  caffein 


i.  Physical 
concentration 


2.  Enrichment 


Examination 

of  water  for 

^ 

typhoid 
bacilli 

3.  Isolation 

4.  Identifica-       ! 

tion 

process 

b.  Jackson's  lactose  bile 

c.  Parietti's  carbol  broth 

a.  Eisner's  gelatin  medium 

b.  Endo's  medium 

c.  Loeffler's  malachite  green 

medium 

d.  Drigalski-Conradi  agar 

e.  Hiss's  medium 
/.  Hesse's  medium 

a.  Morphological  and  cultural 

characters 

b.  Agglutination 


ISOLATION  OF  SPECIFIC  PATHOGENES         87 

Of  the  comparative  advantages  of  these  methods 
it  is  still  too  early  to  speak  with  finality.  Up  to  the 
present  time  the  use  of  caffein  and  lactose  bile  has 
apparently  been  followed  by  the  best  results,  and  it 
seems  likely  that  of  the  precipitation  methods  that 
employing  the  oxychloride  of  iron  is  the  best.  Lubenau 
(Lubenau,  1907)  has  made  some  interesting  com- 
parisons, using  media  containing  malachite  green  and 
caffein  and  caffein  alone,  in  which  the  advantage  is 
decidedly  in  favor  of  the  latter. 

Identification  of  the  Typhoid  Bacillus.  At  the  end 
of  the  process  the  identification  of  the  pure  cultures 
isolated  is  again  subject  to  considerable  uncertainty. 
The  typhoid  bacillus  belongs  to  a  large  group  which 
contains  numerous  varieties  differing  from  each  other 
by  minute  degrees.  The  inability  to  reproduce  the 
disease  by  inoculation  in  available  test  animals  owing 
to  their  natural  immunity  is  a  serious  drawback; 
and  the  specific  biochemical  characters  of  the  organism 
are,  as  it  happens,  mostly  negative  ones,  as  shown  by 
comparison  with  B.  coli,  to  which  it  is  supposed  to  be 
allied. 

COMPARISON  OF  THE  CHARACTERS  OF  B.  COLI  AND 
B.  TYPHI 

(HORROCKS,  IQOl) 
B.  COLI  B.  TYPHI 

(i)  Surface    Colonies,    Gelatin          (i)  Much    thinner   than    those 

Plates. — Thicker,  and  grow  more  of  B.  coli,  and  grow  more  slowly, 

rapidly  than   those  of  B.   typhi.  After   forty-eight   hours'    incuba- 

After  forty-eight  hours' incubation  tion  at  22°  C.   they  are  hardly 

at  22°  C.  they  are  usually  large  visible  to  the  naked  eye. 
and  characteristic. 


ELEMENTS  OF  WATER  BACTERIOLOGY 


(2)  Gelatin-stab. — Quick  growth 
on  the  surface  and  along  the  line 
of  inoculation. 


(2)  Slow  growth  on  the  surface 
like  the  colonies;   along  the  line  of 
inoculation,  the  growth  is  much 
thinner,  and  often  ends  below  in  a 
few  white  points  consisting  of  dis- 
crete colonies. 

(3)  Thin  narrow  grayish-white 
growth,     crenated     margin     not 
marked  as  in  B.  coli. 

(4)  No  formation  of  indol. 

(5)  Unchanged  after  a  month. 

(6)  Very  small  amount  of  acid 
produced,  requiring  not  more  than 
6  per  cent  of   N/io  alkali  to  neu- 
tralize it. 

(7)  No  change. 

(8)  No  gas  formation. 


(3)  Gelatin-slope. — Thick,  broad 
grayish-white  growth  with  a  cre- 
nated margin. 

(4)  Witte's    Peptone   and   Salt 
Solution. — Indol  produced. 

(5)  Milk.— Coagulated. 

(6)  Litmus-whey,  one  week  at 
37°    C.      Acid   produced   usually 
requiring  from  20  to  40  per  cent  of 
N/io  alkali  to  neutralize  it. 

(7)  Neutral-red  Glucose-agar. — 
Marked  green  fluorescence. 

(8)  Glucose-gelatin    and    Lac- 
tose-gelatin Shake  Cultures,  and 
Glucose-agar-stab. — Marked     gas 
formation. 

(9)  Gelatin,  25  oer  cent,  incu- 
bated   at    37°    C.  — Thick    film 
appears  on  the  surface. 

(10)  Potato. — As  a  rule,  a  thick 
yellowish-brown  growth. 

(n)  Proskauer  and  Capaldi's 
Media.  No.  I,  after  twenty  hours' 
growth,  medium  acid.  No  II, 
Growth,  medium  neutral  or  faintly 
alkaline. 

(12)  Nitrate-broth. — Nitrate  re- 
duced to  nitrite. 

(13)  Microscopical         Appear- 
ances.— A    small    bacillus     often 
like  a  coccus,  not  motile  as  a  rule. 

(14)  Flagella. — Usually  i  to  3, 
short  and  brittle;   sometimes  8  to 
12,  long  and  wavy. 

(15)  Agglutination. — As  a  rule, 
no  agglutination  with  a  dilute  anti- 
typhoid serum. 

In  addition  to  the  biochemical  characters  noted  above 
the  typhoid  bacillus  is  characterized  by  its  failure  to 
produce  gas  and  by  its  feeble  acid  production  in  lactose 


(9)  No  film  appears  on  the  sur- 
face, but  a  general  growth  takes 
place  throughout  the  tube. 

(10)  Thin    transparent    growth 
hardly  visible  to  the  naked  eye. 

(u)  No.  I,  no  growth  or  change 
in  the  reaction  of  the  medium. 
No.  II,  Growth,  medium  acid. 


•(i  2)  Reduction  of  nitrate  not  so 
marked. 

(13)  Usually    longer    than    B. 
coli;    highly  motile,  with  a  quick 
serpent-like  movement. 

(14)  Usually  8  to  12,  long  and 
wavy. 

(15)  Marked  agglutination  with 
dilute  anti-typhoid  serum. 


ISOLATION  OF  SPECIFIC  PATHOGENES         89 

media  and  by  its  characteristic  colonies  on  Drigalski- 
Conradi  agar,  Endo  medium,  Hiss  medium,  and  Hesse 
agar.  In  studying  its  immunity  reactions  agglutina- 
tion should  in  all  important  cases  be  supplemented 
by  the  Pfeiffer  reaction  and  the  absorption  test  which 
will  be  found  described  in  standard  text-books  on 
bacteriology. 

Of  the  many  observers  who  have  reported  the  isola- 
tion of  the  typhoid  bacillus  from  water,  all  but  the  most 
recent  are  quite  discredited,  on  account  of  the  insuf- 
ficiency of  the  confirmatory  tests,  and  even  the  latest 
results  should  be  received  with  caution.  Since  the 
introduction  of  the  Widal  (Widal,  1896)  reaction, 
founded  on  the  fact  that  typhoid  bacilli  examined 
under  the  microscope  in  the  diluted  blood-serum  of 
a  typhoid  patient  lose  their  motility  and  "  agglutinate  " 
or  clump  together,  an  important  aid  has  been  fur- 
nished in  the  diagnosis.  Yet  serum  tests  are  notably 
erratic,  and  insufficient  to  identify  an  organism  with- 
out an  exhaustive  study  of  biochemical  reactions. 
Many  organisms  are  agglutinated  by  typhoid  serum  in 
a  more  or  less  dilute  solution,  and  agglutinations  are 
not  significant  unless  obtained  in  dilutions  as  great  as 
1-500  or  i-iooo.  The  discovery  of  the  Bacillus  dysen- 
teriae  of  Shiga,1  which  closely  resembles  the  typhoid 
bacillus,  has  made  the  identification  of  the  latter  more 
dubious  than  ever.  Hiss  (1904)  has  shown  that  the 
fermentation  and  agglutination  reactions  of  the  two 
organisms  are  in  many  respects  alike,  and  Park  and  his 

*  For  an    account    of    biology    of    B.  dysenteriae    the    student    is 
referred  to  an  article  by  Dombrowsky;  1903. 


90         ELEMENTS  OF  WATER  BACTERIOLOGY 

associates  (1904)  have  shown  that  there  are  not  less 
than  three  distinct  types  of  dysentery  bacilli  forming 
that  group. 

Isolation  of  Typhoid  Bacilli  from  Water.  The  methods 
we  have  been  discussing  have  many  of  them  been  used 
chiefly  for  obtaining  the  typhoid  bacillus  from  fasces, 
which  is  much  easier  than  its  isolation  from  polluted 
water.  There  are,  however,  a  number  of  cases  in  which 
the  organism  has  undoubtedly  been  isolated  from 
polluted  water,  as  by  Kubler  and  Neufeld  (Klibler  and 
Neufeld,  1899),  who  examined  a  farmhouse  well  at 
Neumark  in  1899,  and  Fischer  and  Flatau  (Fischer  and 
Flatau,  1901),  who  discovered  an  organism  responding 
to  a  most  exhaustive  series  of  tests  for  the  typhoid 
bacillus  in  a  well  at  Rellingen  in  1901.  In  these  cases 
the  water  was  directly  plated  upon  Eisner's  medium  or 
phenolated  gelatin  with  no  preliminary  process  of 
enrichment.  Willson  (Willson,  1905)  summarized  the 
instances  in  which  the  typhoid  bacillus  had  been 
isolated  from  infected  drinking  water,  up  to  1905,  and 
included,  in  addition  to  the  above-mentioned  cases,  the 
following : 

1.  By  Losener,  in  1895,  from  the  Berlin  water  supply. 

2.  By  Conradi,  in  1902,  from  a  well  at  Pecs  in  Hun- 
gary, by  use  of  carbol  gelatin  plates. 

3.  By   Jaksch   and   Rau,   in    1904,   from   the   water 
supply. of  Prague,  and  also  from  the  river  Moldau,  by 
caffein-nutrose  crystal  violet  agar. 

4.  By  Stroszner,  in  1904,  from  a  well  near  Budapest, 
by  the  same  method. 

Several  other  instances  in  which  the  isolated  organ- 


ISOLATION  OF  SPECIFIC  PATHOGENES         91 

isms  gave  positive  agglutination  tests,  as  well  as  the 
usual  cultural  reactions,  are  also  cited  by  Willson. 

During  the  last  5  years  a  number  of  successful  isola- 
tions of  the  typhoid  bacillus  have  been  reported  in 
America.  An  organism  obtained  from  the  water-supply 
of  Scran  ton,  Pa.,  in  1907,  by  simple  enrichment  in 
Parietti  bouillon,  was  identified  as  the  typhoid  bacillus 
by  Prof.  Fox  after  a  very  careful  series  of  tests  with 
immune  sera  (Pennsylvania,  1908).  The  most  impor- 
tant results  have  been  achieved,  however,  by  Jackson 
with  lactose  bile  enrichment  and  subsequent  plating  on 
Hesse  agar.  He  reports  the  isolation  of  B.  typhi  from 
10  c.c.  samples  of  the  Grass  River  at  Canton,  N.  Y., 
and  of  a  pond  and  stream  at  Hastings,  N.  Y.,  (both 
used  as  sources  of  water-supply)  and  from  two  i  c.c. 
samples  of  the  Hudson  River  near  Hastings  at  the 
time  of  the  typhoid  epidemic  there  (Jackson  and  Melia, 
1909).  Stokes  and  Hachtel  (1910)  by  the  same  method 
found  organisms  corresponding  to  typhoid_in  their  general 
cultural  reactions  in  four  samples  of  surface-waters 
(two  of  them  from  an  impounding  reservoir  of  the 
Baltimore  supply),  in  the  sediment  of  a  school  well 
supposed  to  have  caused  typhoid  fever,  in  a  sewage- 
polluted  stream  and  in  two  samples  of  market  oysters. 
These  organisms  agglutinated  with  the  blood  of  typhoid 
patients  in  1/50  and  i/ioo  dilutions,  but  with  an 
immune  serum  producing  agglutination  with  a  standard 
laboratory  typhoid  culture  in  dilution  of  1/25,000 
these  water  organisms  would  only  agglutinate  in 
dilutions  of  1/250  or  1/500.  Their  identity  must 
therefore  be  regarded  as  somewhat  doubtful.  The 


92         ELEMENTS  OF  WATER  BACTERIOLOGY 

same  authors  (Stokes  and  Hachtel,  1912)  have  more 
recently  reported  the  isolation  of  the  typhoid  bacillus 
from  the  water  in  the  neighborhood  of  a  polluted 
oyster  bed. 

The  search  for  the  typhoid  bacillus  is  usually  sug- 
gested when  an  outbreak  of  the  disease  has  cast  strong 
suspicion  upon  some  definite  source  of  water-supply. 
By  the  time  an  epidemic  manifests  itself,  however, 
the  period  of  the  original  infection  is  long  past,  and  the 
chances  are  good  that  any  of  the  specific  bacilli  once 
present  will  have  disappeared.  While  elaborate  exper- 
iments have  shown  that  B.  typhi  may  persist  in 
sterilized  water  for  upwards  of  2  months  and  in  unster- 
ilized  water  from  3  days  to  several  weeks,  the  number 
of  the  organisms  present  is  always  very  rapidly  reduced. 
Even  in  highly  polluted  water  their  number  is  propor- 
tionately small;  as  is  well  shown  by  the  experiments 
of  Laws  and  Andrewes  (Laws  and  Andrewes,  1894) 
who  entirely  failed  to  isolate  the  typhoid  bacillus  from 
the  sewage  of  London  and  found  only  two  colonies  of 
the  organism  on  a  long  series  of  plates  made  from  the 
sewage  of  a  hospital  containing  forty  typhoid  patients. 
So  Wathelet  (Wathelet,i895)  found  that  of  600  colonies 
isolated  from  typhoid  stools  and  having  the  appearance 
characteristic  of  B.  coli  and  B.  typhi  only  10  belonged 
to  the  latter  species. 

Epidemiological  evidence  confirms  these  results  and 
indicates  that  the  number  of  typhoid  bacilli  even  in 
polluted  water  probably  is  never  very  great,  while  the 
fate  of  Lowell  and  Lawrence  in  1890-91  and  the  more 
recent  epidemics  at  Butler,  Pa.,  and  Ithaca,  N.  Y., 


ISOLATION  OF  SPECIFIC  PATHOGENES          93 

demonstrate  that  even  a  small  number  of  virulent 
organisms  can  bring  about  an  almost  wholesale  infection. 
Indeed,  if  the  virulent  organism  were  as  abundant  as 
some  results  would  indicate  (Remlinger  and  Schneider, 
1897),  the  human  race  would  long  since  have  been 
exterminated.  A  negative  result  in  testing  for  typhoid 
bacilli  has  no  significance  and  there  is  danger  that  it 
may  be  misinterpreted  if  the  fact  that  it  has  been  made 
comes  to  public  knowledge.  In  spite  of  this  danger, 
however,  and  in  spite  of  the  laborious  and  time-con- 
suming nature  of  the  process,  the  increasingly  large 
number  of  positive  isolations  in  recent  years  indicate 
that  it  is  well  worth  trying  in  cases  of  special  importance. 
The  search  for  the  typhoid  bacillus  should  of  course 
never  supersede  the  examination  for  colon  bacilli, 
since  the  latter  are  so  much  more  numerous  in  water 
and  so  much  more  easily  identified.  Because  of  these 
facts,  colon  bacilli  will  continue  to  be  our  best  index 
of  pollution,  while  the  positive  isolations  of  the  typhoid 
bacillus  will  supply  additional  proof  of  the  deadly 
character  of  a  water  containing  it. 

Other  Bacteria  of  the  Typhoid  Group  Related  to 
Intestinal  Disease.  The  typhoid  bacillus  and  the  colon 
bacillus  (which  will  be  fully  discussed  in  succeeding  chap- 
ters) stand  at  the  opposite  ends  of  a  series  of  many  dif- 
ferent varieties  of  organisms  which  are  intermediate  in 
their  properties  between  B.  typhi  and  B.  coli,  all  being 
non-spore-forming,  non-liquefying  rods,  which  produce 
a  more  or  less  characteristic  growth  on  solid  media. 
Durham  (1898)  divided  these  forms  into  three  main 
divisions,  grouped,  respectively,  about  B.  typhi,  B. 


94 


ELEMENTS  OF  WATER  BACTERIOLOGY 


enteritidis  and  B.  coli.  Organisms  of  the  first  division 
ferment  neither  dextrose,  lactose  nor  saccharose;  those 
of  the  second  ferment  dextrose  but  not  lactose;  and 
those  of  the  B.  coli  division  form  gas  in  both  these 
sugars.  The  relationship  of  the  commonest  species 
is  indicated  in  tabular  form  below: 

BACTERIA  OF  THE  COLON-TYPHOID   GROUP 


Species. 

Dextrose. 

Lactose. 

Gas  For- 
mation. 

Acid  Pro- 
duction. 

Gas  For- 
mation. 

Acid  Pro- 
duction. 

B.  alcaligenes  

None 
None 
None 

Active 
Active 
Active 

Active 

None 
Slight 
Distinct 

Strong 
Strong 
Strong 

Strong 

None 
None 
None 

None 
None 
None 

Active 

None 
None 
None 

Slight 
Slight 
Slight 

Strong 

B   typhi 

B   dysenterise     ...        ... 

B   enteritidis 

Paratyphoid  bacilli  

Hog  cholera  bacillus  

B   coli 

In  the  typhoid  division,  B.  alcaligenes  and  B.  dysen- 
teriae are  the  best-known  forms,  besides  B.  typhi  itself. 
B.  alcaligenes  stands  at  the  lower  end  of  the  whole 
series  in  fermentative  power.  B.  typhi  forms  a  slight 
initial  acidity  in  milk  and  a  slight  acidity  in  dextrose 
broth,  while  the  reaction  of  B.  alcaligenes  in  sugar 
media  is  always  alkaline.  B.  dysenteriae,  on  the  other 
hand,  differs  from  B.  typhi  in  the  direction  of  the  B. 
enteritidis  group,  producing  a  well-marked  acid  reac- 
tion, but  no  gas  in  dextrose  media.  B.  typhi  and  B. 
dysenteriae  are,  of  course,  also  distinguished  by  their 
specific  serum  reactions. 


ISOLATION  OF  SPECIFIC  PATHOGENES         95 

The  second  great  division  of  the  colon-typhoid 
bacteria  is  the  hog  cholera  group,  or  the  Gartner  group, 
as  Durham  (1898)  called  it.  As  defined  by  him,  it 
differed  from  the  typhoid  group  by  gas  formation  in 
dextrose,  and  from  the  colon  group  by  the  production 
of  a  final  alkaline  reaction  in  milk.  It  includes  the 
Gartner  bacillus  (B.  enteritidis),  the  hog  cholera  bacillus 
(B.  cholerae  suis),  and  the  paratyphoid  bacilli.  Some 
of  these  forms,  the  paratyphoid  bacilli,  for  example, 
and  B.  enteritidis  (isolated  in  cases  of  meat  poisoning), 
produce  intestinal  disease  in  man. 

There  is  no  doubt  that  water  is  sometimes  the 
means  of  distributing  the  germs  of  dysentery  and 
diarrhoea,  as  shown  by  the  decrease  of  these  diseases 
in  Burlington,  Vt,  (Sedgwick,  1902),  and  other  com- 
munities where  pure  water-supplies  have  been  sub- 
stituted for  polluted  ones.  Thresh  (Thresh,  1903) 
described  an  epidemic  of  over  1000  cases  of  diarrhoea 
with  14  deaths,  which  occurred  in  England  at  Chelmsford 
and  Widford,  and  was  undoubtedly  spread  by  the 
public  water-supply.  A  somewhat  similar  epidemic 
of  dysentery  occurred  in  Warren  and  Kittanning,  in 
Pennsylvania,  in  1906,  which  was  unquestionably  due 
to  contamination  of  the  water,  in  this  case  a  river- 
supply.  It  is  possible  that  the  examination  of  water 
for  the  B.  dysenteriae  may  in  the  future ."!  help  to 
throw  important  light  on  the  sanitary  condition  of 
a  water. 

Starkey  (1909  and  1911)  believes  that  all  organisms 
giving  the  general  reactions  of  the  Gartner  and  para- 
typhoid groups  are  significant  and  warrant  the  con- 


96         ELEMENTS  OF  WATER  BACTERIOLOGY 

demnation  of  a  water-supply.  The  difficulty,  however, 
is  that  while  non-acid-forming  bacteria  of  this  general 
type  are  sometimes  found  in  faeces,  they  are  also 
found  in  other  habitats,  and  they  are  less  abundant 
proportionately,  in  polluted  than  in  stored  and  safer 
waters.  If  true  dysentery  and  paratyphoid  bacilli 
can  be  isolated  and  identified  by  serum  reactions  it  is, 
of  course,  highly  important.  Houston' (1911),  however, 
has  recently  tested  the  method  suggested  by  Starkey 
(1906)  for  isolating  these  forms  and  found  that  it  gave 
negative  results  even  with  a  water  artificially  infected 
with  about  14  typhoid  bacilli  and  21  Gartner  bacilli 
per  c.c.  In  his  own  studies  Houston  reports  that  in 
the  examination  of  13,442  microbes  from  polluted 
river  water  he  found  only  one  member  of  the  Gartner 
group;  and  in  another  study  of  20,771  colonies  he 
found  only  2  typhoid-like  forms. 

Isolation  of  the  Cholera  spirillum.  The  isolation  of 
the  cholera  spirillum  from  water  can  probably  be  accom- 
plished with  somewhat  less  difficulty  than  is  encoun- 
tered in  the  case  of  B.  typhi.  Schottelius  (Schottelius, 
1885)  was  the  first  to  point  out  the  necessity  for  grow- 
ing this  organism  in  an  alkaline  medium,  and  Loeffler 
(Loeffler,  1893)  found  that  its  isolation  from  water 
could  be  successfully  accomplished  by_adding  10  c.c. 
of  alkaline  pepton  broth  to  200  c.c.  of  the  infected 
water  and  incubating  for  24  hours  at  37  degrees,  when 
the  organism  could  be  found  at  the  surface  of  the 
medium. 

Somewhat  earlier  than  this  Dunham  (Dunham,  1887) 
had  made  a  special  study  of  the  chemical  reactions  of 


ISOLATION  OF  SPECIFIC  PATHOGENES         97 

the  cholera  spirillum  and  found  that  the  organism  would 
grow  abundantly  in  a  solution  containing  i  per  cent 
peptone  and  0.5  per  cent  salt  .(Dunham's  solution), 
producing  the  "  cholera-red  or  nitroso-indol  reaction." 
This  medium  was  brought  into  practical  use  by  Dunbar 
(Dunbar,  1892),  who  succeeded  in  isolating  the  organisms 
from  the  water  of  the  Elbe  in  1892,  during  the  cholera 
epidemic  at  Hamburg. 

Koch  (Koch,  1893)  prescribed  the  following  method 
for  the  isolation  of  the  organism  from  water: 

To  100  c.c.  of  the  water  to  be  examined  is  added  i 
per  cent  pepton  and  i  per  cent  salt.  The  mixture  is 
then  incubated  at  37  degrees.  After  intervals  of  10, 
15,  and  20  hours  the  solution  is  examined  microscopically 
for  comma-shaped  organisms,  and  agar  plate  cultures 
are  made  which  are  likewise  incubated  at  37  degrees. 
If  any  colonies  showing  the  characteristic  appearance 
of  the  cholera  spirillum  are  found,  these  are  examined 
microscopically,  and  if  comma-shaped  organisms  are 
present,  inoculations  are  made  into  fresh  tubes  to  be 
further  tested  by  means  of  the  indol  reaction  and  by 
inoculation  into  animals. 

Stokes  and  Hachtel  (1912)  have  suggested  the  use 
of  a  modified  Hesse  agar  containing  starch  for  the 
isolation  of  the  cholera  spirilla,  which  produce  acid  on 
such  a  medium,  while  the  colon-typhoid  organisms  do 
not.  The  glycerin  and  lactose  are  omitted  from  the 
medium  described  on  p.  79  and  10  gms.  of  soluble 
starch  are  added.  The  intestinal  spirilla  as  a  class 
form  round,  spreading,  pinkish  colonies  on  the  starch 
medium,  while  colonies  of  other  intestinal  bacilli  remain 


98         ELEMENTS  OF  WATER  BACTERIOLOGY 

blue.     The  medium  is  best  used  after  the  Koch  enrich- 
ment method  described  above. 

Other  pathogenic  organisms  have  been  isolated  from 
waters,  according  to  the  accounts  of  numerous  investi- 
gators, but  from  the  sanitary  point  of  view  the  typhoid 
and  cholera  bacilli  are  of  most  importance,  since  these 
are  manifestly  the  germs  of  disease  most  likely  to  be 
disseminated  through  this  medium.  For  the  detection 
of  B.  anthracis  and  other  spore-forming  pathogenic 
bacteria  which  may  at  times  gain  access  to  water  from 
stockyards,  slaughter-houses,  etc.,  the  method  suggested 
by  Frankland  (Frankland,  1894)  may  be  adopted. 
The  water  to  be  examined  is  heated  to  90  degrees  for 
2  minutes  and  then  plated,  the  characteristic  colonies 
of  the  anthrax  organism  being  much  more  easily  dis- 
cerned after  the  destruction  of  tne  numerous  non- 
sporing  water  bacteria. 


CHAPTER  VI 

THE  COLON  GROUP  OF  BACILLI  AND  METHODS  FOR 
THEIR  ISOLATION 

The  Colon  Group  of  Bacilli.  The  Bacillus  coli  was 
first  isolated  by  Escherich  (Escherich,  1884)  from  the 
faeces  of  a  cholera  patient.  It  was  subsequently  found 
to  be  a  normal  inhabitant  of  the  intestinal  tract  of  man 
and  many  other  animals,  and  to  occur  regularly  in 
their  excreta,  and  on  this  account  it  became  of  the 
highest  interest  and  importance  to  sanitarians,  since 
its  presence  in  water-supplies  was  regarded  as  direct 
evidence  of  sewage  pollution. 

Specific  disease  germs  are  difficult  to  isolate  even 
when  they  are  present;  and  water  may  of  course  be 
grossly  polluted  with  sewage  without  any  specific 
disease  germs  being  there  at  all.  All  sewage-polluted 
water,  however,  is  potentially  dangerous,  since  where 
faecal  matter  exists,  disease  germs  are  at  any  time  likely 
to  appear.  A  test  for  faecal  material  as  distinguished 
from  infected  material  is,  therefore,  essential;  and 
for  such  a  test  the  colon  group  of  bacilli  are  specially 
well  suited.  They  are  not  dangerous  in  themselves, 
but  they  are  significant  as  indices  'of  the  probable 
presence  of  disease  germs. 

The  so-called  Bacillus  coli  may  be  described  as  a  short, 

99 


100       ELEMENTS  OF  WATER  BACTERIOLOGY 

usually  motile  rod,  with  diameter  generally  less  than  one 
micron  and  exhibiting  no  spore  formation.  It  often  ap- 
pears in  pairs  of  rods  so  short  as  to  suggest  a  diplococcus. 
It  decolorizes  by  the  Gram  stain.  It  forms  thin,  irregu- 
lar translucent  films  upon  the  surface  of  gelatin,  called 
"  grape-leaf  colonies "  by  the  Germans,  produces 
no  liquefaction,  and  gives  a  wire-nail-like  growth  in 
stick  cultures.  It  forms  a  white  translucent  layer  of 
characteristic  appearance  upon  agar,  produces  a  more 
or  less  abundant,  moist,  yellowish  growth  on  potato, 
and  turbidity  and  some  sediment  in  broth;  it  ferments 
dextrose  and  lactose  with  the  formation  of  gas  of  which 

the  ratio  is  approximately,  -  -  =  - ,  as  ordinarily  deter- 

C(J2         I 

mined;  a  strong  acid  reaction  and  gas  are  produced 
in  many  other  sugar-containing  media.  The  organism 
generally  gives  a  characteristic  reaction  in  esculin 
media  and  typically  reduces  neutral  red,  changing 
its  color  to  canary  yellow  with  a  greenish  fluorescence. 
It  grows  in  the  Capaldi-Proskauer  media,  forming 
acid  in  the  albumin-free  medium,  No.  i,  and  giving 
a  neutral  or  alkaline  reaction  in  the  peptone-mannite 
medium  No.  2.  It  coagulates  casein  in  litmus  milk, 
and  reduces  the  litmus  with  subsequent  slow  return 
of  the  color  (red),  and  generally  forms  indol  in  peptone 
solution.  Many  cultures  of  this  organism  are  fatal 
to  guinea  pigs  when  the  latter  are  inoculated  sub- 
cutaneously  with  one-half  c.c.  of  a  24-hour  bouillon 
culture,  and  most  cultures  produce  death  when  this 
amount  is  inoculated  intraperitoneally.  Although  not 
a  spore-forming  bacillus,  and  in  general  not  possessing 


THE  COLON  GROUP  OF  BACILLI      101 

great  resistance  against  antiseptic  substances,  B.  coli 
is  less  susceptible  to  phenol  than  are  many  other  forms, 
especially  certain  water-bacteria. 

We  have  spoken  as  if  Bacillus  coli  were  a  single  defi- 
nite organism.  As  a  matter  of  fact  it  is  a  name  applied 
to  a  considerable  group  of  distinct  forms  which  may 
be  split  up  almost  as  far  as  one  wishes  by  the  applica- 
tion of  various  biochemical  tests.  The  "  colon  bacillus," 
as  we  have  pointed  out,  usually  does  not  liquefy  gelatin 
and  reduces  neutral  red  and  coagulates  milk,  and 
produces  indol;  but  there  are  closely  allied  forms  which 
differ  from  the  type  in  one  or  more  of  these  respects. 
The  colon  group,  as  Smith  (1893)  long  ago  pointed 
out,  may  first  be  divided  into  two  distinct  subtypes 
according  to  the  action  of  the  organisms  upon  saccharose. 
One  subtype  forms  gas  and  acid  in  saccharose  media 
and  the  other  does  not.  Winslow  and  Walker  (1907) 
have  found  that  those  strains  which  ferment  saccharose 
attack  raffinose  also,  and  point  out  that  these  two 
sugars  which  behave  alike  are  those  which  lack  the 
aldehyde  grouping  characteristic  of  dextrose  and  lactose. 
The  application  of  tests  in  other  carbohydrate  media, 
such  as  dulcite,  adonite,  inulin,  etc.,  make  it  possible 
to  recognize  perhaps  a  hundred  distinct  types  each 
characterized  by  a  particular  combination  of  reactions. 

The  results  obtained  by  the  "  colon  test  "  will  of 
course  depend  largely  upon  the  definition  of  what  a 
colon  bacillus  is;  and  there  is  marked  disagreement 
upon  this  point  among  different  observers.  Konrich 
(1910)  tabulates  the  tests  used  by  34  different  workers, 
All  of  them  defined  the  colon  bacillus  as  a  Gram- 


102       ELEMENTS  OF  WATER  BACTERIOLOGY 

negative  non-spore-forming  rod,  but  there  was  unanimity 
in  no  other  respect;  26  of  the  34  included  the  formation 
of  acid  and  gas  in  dextrose  media,  22  the  coagulation 
of  milk,  21  the  formation  of  indol,  18  the  formation 
of  acid  and  gas  in  lactose  media,  and  18  the  failure  to 
liquefy  gelatin.  No  other  test  was  used  by  more  than 
13  out  of  the  34  observers.  Konrich  (1910)  himself 
found  that  of  over  600  colon-like  organisms  from  faeces, 
all  produced  gas  in  dextrose  broth,  79  per  cent  formed 
acid  and  77  per  cent  gas  in  lactose  broth,  65  per  cent 
coagulated  milk,  59  per  cent  fermented  dextrose  at 
46°,  54  per  cent  reduced  neutral  red,  38  per  cent  formed 
indol. 

Ferreira,  Horta  and  Paredes  (i9o8a)  studied  117 
strains  of  lactose-fermenting  bacilli  from  human  faeces. 
All  proved  to  be  motile  and  Gram  negative,  all  coag- 
ulated milk  and  produced  fluorescence  in  neutral  red 
media,  none  liquefied  gelatin  in  15  days,  all  but  one 
formed  indol.  Dextrose,  lactose,  maltose,  galactose, 
and  mannit  were  fermented  by  almost  all  strains, 
while  gas  was  formed  in  saccharose  by  38  per  cent, 
in  dulcite  by  69  per  cent  and  in  inulin  by  12  per  cent 
of  the  strains  studied.  The  "  Proskauer  reaction " 
(apparently  the  Voges-Proskauer  reaction,  though  it 
is  not  quite  clearly  stated)  was  positive  only  8  times 
out  of  117  strains  in  dextrose,  only  7  times  (out  of  48 
strains  tested)  in  galactose,  and  not  at  all  in  dulcite  or 
inulin;  lactose  and  maltose,  on  the  other  hand,  showed 
it  in  almost  every  case.  Copeland  and  Hoover  (1911) 
report  that  out  of  3000  colon-like  organisms  fermenting 
-lactose  bile  65  per  cent  gave  positive  tests  for  B.  coli 


THE  COLON  GROUP  OF  BACILLI  103 

in  milk,  nitrate  solution,  pepton  solution  and  gelatin; 
28  per  cent  failed  to  produce  indol  and  5  per  cent  did 
not  reduce  nitrates.  Numerous  other  results  indicat- 
ing similar  variations  are  cited  in  Chapters  VII  and 
VIII. 

Where  shall  the  line  be  drawn?  The  English  bac- 
teriologists usually  require  in  addition  to  the  morpholog- 
ical characters  mentioned  above,  motility,  non-liquefac- 
tion of  gelatin,  fermentation  of  dextrose  and  lactose 
media,  coagulation  of  milk,  production  of  indol,  and 
reduction  of  neutral  red.  The  usual  American  pro- 
cedure has  included  reactions  in  dextrose  broth,  milk, 
peptone  solution  (for  indol) ,  gelatin  (absence  of  liquefac- 
tion), and  reduction  of  nitrates.  Of  late  years,  how- 
ever, there  has  been  a  growing  feeling  that  such 
arbitrary  definitions  went  either  too  far  or  not  far 
enough.  The  whole  group  of  lactose-fermenting  bacilli 
is  characteristically  of  intestinal  origin.  That  we  be- 
lieve to  have  been  clearly  established  by  results  to 
be  cited  later  in  this  chapter  and  in  the  succeeding  one. 
A  differentiation  between  various  sub-types  of  this 
group  can  only  be  properly  justified  by  the  fact  that 
some  of  them  are  less  resistant  in  water  than  others, 
and  hence  are  indicative  of  fresh  and  recent  pollution. 
There  is  some  evidence  that  such  is  the  case  which  will 
be  discussed  in  Chapter  VIII,  but  whatever  may  be 
concluded  from  the  somewhat  conflicting  opinions 
on  this  point  it  appears  certain  that,  in  temperate 
climates  at  least,  the  whole  class  of  lactose  fermenters 
should  be  absent  from  safe  water  supplies.  As  the 
Committee  on  Standard  Methods  of  Water  Analysis 


104       ELEMENTS  OF  WATER  BACTERIOLOGY 

(1912)  wisely  concludes,  "  The  entire  group  is  typical 
of  the  presence  of  faecal  matter  when  water  or  sewage 
examinations  are  to  be  considered."  The  Committee 
defines  the  group  as  a  whole  by  the  following  character- 
istics: "  Fermentation  of  dextrose  and  lactose  with 
gas  production,  short  bacillus  with  rounded  ends, 
non-spore-forming,  facultative  anaerobe,  gives  positive 
test  with  esculin,  grows  at  20°  on  gelatin  and  at  37° 
on  agar,  non-liquefying  in  14  days  on  gelatin.  Gram- 
staining  negative."  This  definition  again,  however, 
includes  debatable  elements.  It  seems  to  us  very 
doubtful  whether  there  is  sufficient  evidence  to  warrant 
making  the  esculin  reaction  a  general  criterion  of  the 
colon  group;  and  bacteria  which  liquefy  gelatin  more 
or  less  slowly  grade  into  otherwise  identical  non- 
liquefying  forms  by  almost  imperceptible  degrees. 

We  hesitate  to  add  another  to  the  long  list  of  arbitrary 
definitions  of  the  much-defined  colon  bacillus;  but  it 
does  seem  to  us  important  to  get  down  to  rock  bottom. 
From  this  standpoint  we  believe  that  the  colon  group 
may  be  defined  as  including  all  aerobic  non-spore- 
forming  bacilli  which  produce  acid  and  gas  in  dextrose 
and  lactose  media.  For  practical  purposes  the  test 
may  be  further  reduced  to  positive  reactions  in  a  lac- 
tose fermentation  medium,  growth  on  an  aerobic  agar 
streak,  and  microscopic  examination,  since  almost  all 
forms  which  ferment  lactose  ferment  dextrose  as  well. 
The  relative  importance  of  the  various  subdivisions  of 
this  general  group  will  be  discussed  in  Chapter  VIII. 

Isolation  of  Colon  Bacilli  by  Direct  Plating.  The  Wurtz 
litmus-lactose-agar  plate  (Wurtz,  1892),  as  noted  in 


THE  COLON  GROUP  OF  BACILLI  105 

Chapter  IV,  furnishes  one  ready  method  for  the  isola- 
tion of  B.  coli  from  water,  and  it  was  used  by  Sedgwick 
and  Mathews  for  the  purpose  as  early  as  1892  (Mathews, 
1893).  The  process  is  based  upon  the  fact  already 
alluded  to,  that  B.  coli  readily  ferments  lactose  with 
the  formation  of  acid.  If,  therefore,  plates  are  made 
with  agar  containing  both  lactose  and  litmus,  the  colon 
colonies  develop  as  red  spots  in  a  blue  field.  Since 
organisms  other  than  B.  coli  (notably  the  streptococci) 
may  also  develop  red  colonies,  it  is  necessary  to  examine 
them  further.  This  is  done  by  fishing  from  isolated 
colonies,  replating  and  inoculating  into  other  media 
for  identification. 

The  plate  method  of  isolation  is  recommended  by  the 
Committee  on  Standard  Methods  of  Water  Analysis 
(1912)  for  sewages  and  polluted  waters,  in  which  colon 
bacilli  are  present  in  i  c.c.  or  less.  They  recommend 
that  Petri  dishes  with  porous  covers  be  used  and  that 
incubation  be  carried  out  at  40°  instead  of  37°.  For 
success  in  the  use  of  this  method  it  is  necessary  to 
get  a  sufficient  dilution  so  that  colonies  may  be  well 
isolated,  and  to  this  end  it  is  advisable  that  a  number 
of  different  dilutions  be  employed,  a  series  of  plates 
being  prepared  from  each.  Under  any  conditions  the 
detection  of  the  colon  bacillus  is  seriously  hampered 
by  the  development  of  other  forms.  Certain  observers 
have  therefore  added  phenol  to  the  agar  medium,  com- 
bining the  effect  of  high  temperature  and  an  antiseptic 
to  check  the  growth  of  water-bacteria.  Copeland  for 
this  purpose  added  to  his  tubes  .2  c.c.  of  a  2  per  cent 
solution  of  phenol  (Copeland,  1901).  Chick  (Chick, 


106       ELEMENTS  OF  WATER  BACTERIOLOGY 

igoo)  found  that  1.33  parts  of  phenol  in  1000  materially 
decreased  the  number  of  colon  bacilli  which  would 
develop,  while  i  part  gave  very  satisfactory  results, 
the  plates  showing  pure  cultures  of  B.  coli.  The  addi- 
tion of  antiseptics  in  this  way  is  always  open  to  the 
objection  that  weaker  strains  may  be  killed  and  lost. 

In  Germany  the  Endo  medium  and  the  Conradi- 
Drigalski  medium  have  been  extensively  used  for  the 
direct  isolation  of  colon  bacilli  with  excellent  results. 
The  composition  and  use  of  these  media  have  been 
discussed  in  Chapter  V.  It  does  not  appear  that  they 
have  sufficient  advantages  to  compensate  for  the  dif- 
ficulty in  preparing  them. 

The  Use  of  Preliminary  Enrichment  Media  in  the 
Isolation  of  Colon  Bacilli.  The  test  for  the  colon 
bacillus  may  be  made  more  delicate  by  a  preliminary 
cultivation  of  the  sample  in  a  liquid  medium  for  24 
hours  at  37°,  thus  greatly  increasing  the  propor- 
tion of  these  organisms  present  before  plating.  As 
suggested  in  the  classic  researches  of  Theobald  Smith 
(Smith,  1892),  this  method  may  be  made  approx- 
imately quantitative  by  the  inoculation  of  a  series 
of  tubes  with  measured  portions  of  the  water.  If, 
for  example,  of  ten  tubes  inoculated  each  with  yiir 
of  a  cubic  centimeter,  four  show  B.  coli,  we  may 
assume  that  some  40  of  these  organisms  were  present 
in  the  cubic  centimeter.  Irons  (Irons,  1901),  in  a 
comparative  study  of  various  methods  for  the  isola- 
tion of  B.  coli,  showed  that  the  preliminary  enrichment 
frequently  gave  positive  results  when  the  results  of 
the  direct  use  of  the  agar  plate  were  negative,  and 


THE  COLON  GROUP  OF  BACILLI  107 

concluded  that  "  where  the  amount  of  B.  coli  is  small 
and  the  colony  count  large,  the  lactose  plate  for  plating 
water  direct  is  inferior  to  the  dextrose  fermentation- 
tube."  Gage  came  to  a  similar  conclusion  (Gage,  1902). 

The  medium  most  commonly  used  in  the  United 
States  prior  to  1906  for  preliminary  enrichment  was 
ordinary  broth  to  which  i.o  per  cent  of  dextrose  had 
been  added,  and  the  reaction  brought  to  the  neutral 
point.  Into  each  of  a  number  of  fermentation-tubes 
of  this  medium  a  measured  quantity  of  the  water  to  be 
examined  is  inoculated,  and  the  culture  is  incubated  for 
24  hours  at  37.5°  C.  It  used  to  be  customary  to  incu- 
bate for  48  hours.  Recent  experience  has,  however, 
shown  that  a  24-hour  period  gives  approximately  the 
same  results  if  the  production  of  gas  rather  than  any 
specified  amount  of  gas  is  the  criterion  of  a  positive 
test.  Longley  and  Baton  (1907)  found  that  of  1091 
enrichment  tubes  giving  positive  tests  after  48  hours 
only  173  showed  no  gas  in  24  hours;  of  these  latter 
only  two  contained  B.  coli.  The  advantage  of  saving  a 
day  is  so  great  as  to  warrant  the  adoption  of  the  shorter 
period.  At  the  end  of  24  hours  at  least,  the  tubes 
are  examined  for  gas  formation.  If  gas  is  found,  a 
small  amount  of  the  culture  should  be  added,  after 
suitable  dilution,  to  litmus  lactose  agar  and  plated. 

With  polluted  waters  it  will  be  found  advantageous  to 
plate  out  on  the  first  appearance  of  gas  (4-8  hours). 
It  has  been  shown  by  one  of  us  (Prescott,  i902b)  that  a 
very  rapid  development  of  B.  coli  takes  place  in  the 
first  few  hours  after  dextrose  solutions  are  inoculated 
with  intestinal  material,  and  a  nearly  pure  growth  of 


108        ELEMENTS  OF  WATER  BACTERIOLOGY 

colon  bacilli  often  results,  while  other  bacteria  multiply 
more  slowly.  With  highly  polluted  waters  gas  forma- 
tion will  probably  begin  within  12  hours,  but  with  fewer 
colon  bacilli  present  the  duration  must  be  increased. 
If  the  period  of  incubation  be  too  long  continued, 
trouble  in  the  subsequent  steps  of  the  isolation  may  be 
encountered  because  of  overgrowths  by  the  sewage 
streptococci,  or  other  forms  which  check  the  growth 
of  the  colon  bacilli  in  the  later  stages  of  fermentation 
and  finally  kill  them  out.  Even  with  pure  cultures  of 
colon  bacilli  Clemesha  (i9i2b)  has  shown  that  sugar- 
broth  tubes  may  be  almost  sterile  after  4  days. 

When  it  is  desired  to  examine  samples  larger  than 
i  c.c.  it  becomes  necessary  to  modify  the  enrichment 
process  by  adding  the  nutrient  material  to  the  water 
instead  of  the  reverse.  For  this  purpose  dextrose 
broth  or  phenol-dextrose  broth  (consisting  of  broth 
with  10  per  cent  dextrose,  5  per  cent  peptone,  and 
.25  per  cent  phenol)  may  be  added  to  the  sample  of 
water  to  be  enriched  as  suggested  by  Gage  (Gage,  1901). 
Generally  10  c.c.  of  the  broth  is  added  to  100  c.c.  of  the 
water.  The  sample  is  then  incubated  at  37°  for  24  hours, 
and  if  at  the  end  of  that  time  growth  has  taken  place,  a 
cubic  centimeter  is  inoculated  into  a  dextrose  tube. 

Advantages  and  Disadvantages  of  the  Dextrose  Broth 
Fermentation  Tube.  Experience  with  the  dextrose 
broth  fermentation  tube  as  a  first  step  in  the  isola- 
tion of  colon  bacilli  soon  led  to  the  conclusion  that  a 
fair  idea  of  the  sanitary  quality  of  water  could  be 
obtained  from  the  results  of  this  test  taken  by  them- 
selves and  without  the  further  process  of  isolating 


THE  COLON  GROUP  OF  BACILLI  109 

specific  cultures.  It  appeared  that  a  rather  definite 
proportion  of  tubes  showing  a  characteristic  fermenta- 
tion proved  on  further  examination  to  contain  bacilli 
of  the  colon  group;  and  it  was  therefore  suggested 
that  the  dextrose  broth  test  alone  might  be  used  as  a 
rapid  "  presumptive  "  test.  The  underlying  principle 
of  this  method  is  that  B.  coli  develops  rapidly*  in  dex- 
trose broth  with  gas  formation  of  from  25  to  70  per 
cent  of  the  capacity  of  the  closed  arm  of  the  fermenta- 
tion tube.  Of  this  gas  approximately  one-third  is 
carbon  dioxide  and  two-thirds  hydrogen,  that  is,  as  the 

gas  formula  is  generally  expressed,  — —  =  — . 

C(J2         I 

In  testing  a  water  by  this  method  a  series  of  samples, 
in  suitable  dilution,  .001,  .01,  .1,  i.o,  or  10  c.c.,  are  added 
directly  to  the  dextrose-broth  tubes  and  incubated  for 
24  hours  at  37°. 

On  measurement  of  the  gas,  if  the  results  above  given 
are  obtained,  the  reaction  is  considered  typical.  If  the 
amount  of  gas  is  between  10  and  25  percent  or  more  than 
70  per  cent,  or  the  percentage  of  carbon  dioxide  is  greater 
than  40,  the  reaction  is  considered  atypical.  If  no  gas 
forms,  or  less  than  10  per  cent,  the  test  is  called  negative. 

In  recent  years.  Irons  (Irons,  1901)  was  perhaps  the 
first  to  call  attention  to  the  value  of  this  method, 
stating  that  "  when  the  dextrose  tube  yields  approx- 
imately 33  per  cent  of  CO2,  Bacillus  coli  communis  is 
almost  invariably  present."  In  the  next  year  the 
reliability  of  the  fermentation  test  as  an  indication  of 
B.  coli  was  worked  out  by  Gage  (Gage,  1902)  as  given 
in  the  table  on  p.  no: 


110       ELEMENTS  OF  WATER  BACTERIOLOGY 


I  C.C. 

too  c.c. 

Number  of  samples  tested  . 

ei  72 

I  27  C 

Number  giving  preliminary  fermentation  
Per  cent  of  latter  proved  to  contain  coli 

1036 
7O 

474 

71 

Whipple  (Whipple,  1903)  examined  a  large  number 
of  surface-water  supplies  by  this  "  presumptive  test  " 
and  obtained  striking  results,  shown  in  the  following 
table.  The  waters  are  arranged  in  six  groups  according 
to  the  results  of  sanitary  inspection,  group  I  including 
waters  collected  from  almost  uninhabited  watersheds, 
and  group  VI  waters  too  much  polluted  to  be  safely 
used  for  domestic  purposes. 

PERCENTAGE  OF  SAMPLES  OF  WATERS  OF  VARIOUS 
SANITARY  GRADES  GIVING  POSITIVE  TESTS  FOR  B. 
COLI  WHEN  DIFFERENT  AMOUNTS  WERE  EXAMINED 

(WHIPPLE,  1903) 


Group. 

O.I  C.C. 

1.0  C.C. 

10  C.C. 

ioo  c.c. 

500  c.c. 

I  
II 

0.0 

r    o 

3-5 

7    3 

20.8 
ICO 

50.0 
60  o 

50.0 

60  o 

III 

o  o 

7  o 

cJO   O 

so  o 

60  o 

IV  

4.0 

6.8 

41  .  7 

67  .0 

75  -° 

V  

5.0 

13.0 

7<.o 

IOO.O 

IOO.O 

VI 

?  o 

2O    2 

/o 

7S    O 

80  o 

IOO   O 

In  view  of  these  results  Whipple  suggested  the  fol- 
lowing provisional  scheme  of  interpretation: 


Presumptive  Test  for  Bacillus  Coli. 


O.OI  C.C. 

O.I  C.C. 

I.O  C.C. 

10.  0  C.C. 

100  C.C. 

Safe 

o 

o 

o 

o 

-f 

Reasonably  safe  
Questionable      

o 
o 

o 
o 

o 

+ 

+ 
+ 

+ 

+ 

Probably  unsafe. 

o 

+ 

4- 

4- 

4- 

Unsafe                  

-f 

+ 

4- 

4- 

+ 

THE  COLON  GROUP  OF  BACILLI      111 

It  is  undoubtedly  true  that  a  negative  presumptive 
test  is  generally  obtained  with  unpolluted  waters.  For 
example,  in  a  study  previously  cited,  Winslow  and 
Nibecker  (1903)  reported  that  of  775  dextrose-broth 
tubes  inoculated  from  259  unpolluted  sources  only  41 
showed  gas.  On  the  other  hand,  it  is  equally  true 
that  in  a  large  proportion  of  cases  colon  bacilli  are 
isolated  from  positive  dextrose-broth  tubes.  Longley 
and  Baton  (1907)  in  the  examination  of  3553  samples 
of  Potomac  water  obtained  positive  tests  794  times, 
while  B.  coli  was  actually  present  529  times;  67  per 
cent  of  the  presumptive  tests  were  therefore  correct. 
Gage  (1902),  in  the  Massachusetts  work  cited  above, 
found  that  70  per  cent  of  his  fermented  dextrose  tubes 
contained  B.  coli. 

The  work  of  recent  years  has  made  it  clear,  however, 
that  both  the  coincidence  of  negative  presumptive  tests 
with  the  absence  of  B.  coli  and  the  general  coinci- 
dence of  positive  presumptive  tests  with  the  presence 
of  B.  coli,  are  open  to  disastrous  exceptions. 

The  errors  in  the  dextrose  broth  test  are  both  positive 
and  negative ;  it  may  lead  to  the  inference  that  bacteria 
of  the  colon  group  are  present  when  they  are  not, 
and  it  may  fail  to  show  them  when  they  are  really  there. 
In  the  first  place,  with  some  waters,  positive  presump- 
tive tests  may  be  obtained  when  colon  bacilli  are  not 
present.  According  to  Clark  and  Gage  (1903)  there 
are  58  well-described  species  of  bacteria  which  give 
the  presumptive  test  in  dextrose-broth,  of  which  23 
are  widely  separated  from  the  B.  coli  group.  An 
unpublished  investigation  by  Winslow  and  Phelps 


112       ELEMENTS  OF  WATER  BACTERIOLOGY 


indicates  that  the  result  of  the  dextrose  broth  test  is 
markedly  influenced  by  the  factor  of  temperature.  Their 
work  consisted  in  the  examination  of  185  samples  of 
water  from  90  different  sources,  ponds,  brooks,  pools, 
wells  and  springs  in  five  different  States,  Maine,  New 
Hampshire,  Massachusetts,  Michigan  and  Virginia,  at 
three  different  seasons  of  the  year.  All  the  waters 
examined  were,  as  far  as  could  be  determined,  free 
from  specific  pollution,  although  washings  from  roads 
or  pastureland  might  have  had  access  to  some  of  them. 
Most  of  the  sources  were  undoubtedly  unpolluted  and 
the  examination  of  119  samples  for  B.  coli  yielded  only 
12  positive  results.  The  presumptive  test,  however, 
was  obtained  in  a  large  proportion  of  the  cases,  and 
much  more  often  in  summer  than  in  winter  or  spring, 
as  indicated  in  the  table  below. 

DEXTROSE  BROTH  FERMENTATION  IN   185   SAMPLES  OF 
NORMAL  WATERS  AT   DIFFERENT   SEASONS 

(WINSLOW  AND  PHELPS) 
Percentage  of  Positive  Results 


Summer, 
1906. 

Winter. 

Spring. 

Summer, 
1907. 

Framingham,  Mass.  .  .  . 

8? 

62 

23 

57 

Ann  Arbor,  Mich  

95 

47 

Exeter,  N.  H  

82 

IO 

44 

50 

Richmond,  Va  

14 

14 

Mt.  Desert,  Me  

95 

All  stations  

QI 

-27 

2< 

C4 

The  Ann  Arbor  waters  in  this  series  included  a  number 
of  driven  wells  and  the  Mt.  Desert  sources  were  mountain 
brooks  and  ponds  of  the  highest  sanitary  quality. 


THE  COLON  GROUP  OF  BACILLI      113 

Fromme  (1910)  in  this  connection  reports  the  results 
of  673  colon  tests  made  on  the  water  of  the  Elbe  during 
a  period  of  a  year  and  a  half.  We  have  calculated 
from  his  figures  the  average  results  for  the  winter 
months  and  the  summer  months  in  the  table  below. 
It  is  evident  that  typical  colon  bacilli  are  nearly  twice 
as  numerous  in  the  cold  weather  (for  reasons  discussed 
in  Chapter  I)  while  organisms  fermenting  dextrose 
broth  but  proving  not  to  be  B.  coli  are  absolutely 
more  abundant  and  relatively  much  more  abundant 
in  summer. 

GAS   PRODUCERS  AND   B.   COLI  IN   ELBE  WATER 

(AFTER  FROMME,  1910) 


Positive  Dextrose 
Broth  Tests. 

B.  Coli 
Isolations. 

Per  Cent  of  Dex- 
trose Broth  Tests 
Showing  B.  Coli. 

October-March  
April-September.  .  .  . 

415 

258 

363 
170 

8? 
66 

Phelps  and  Hammond  (1909)  cite  a  very  interesting 
case  of  the  same  phenomenon  in  the  case  of  a  ground 
water.  A  deep  well  at  a  hospital  in  Trenton,  N.  J. 
was  temporarily  polluted  from  a  leaking  sewer  and  after 
the  source  of  pollution  had  been  removed  the  condi- 
tion of  the  water  was  carefully  studied  for  a  period  of 
two  months.  During  the  period  between  Sept.  10  and 
Oct.  12  (the  pollution  being  removed  on  Sept.  19) 
of  107  dextrose-fermenting  microbes  isolated  40  failed 
to  produce  gas  in  lactose  broth;  during  the  period 
between  Oct.  12  and  Nov.  9,  52  out  of  64  dextrose-fer- 
menting microbes  failed  to  give  gas  in  lactose  broth. 
All  through  the  investigation  organisms  of  low  fer- 


114       ELEMENTS  OF  WATER  BACTERIOLOGY 


mentative  power,  many  of  them  liquefying  gelatin, 
were ,  present,  but  their  numbers  relatively  increased 
during  the  period  after  the  original  pollution  had  been 
removed. 

Much  valuable  light  is  thrown  upon  the  significance 
of  these  positive  dextrose  broth  results  in  the  absence 
of  the  colon  group  by  the  investigations  of  Clemesha 
(igi2a).  In  a  careful  study  of  46  samples  of  human 
faeces  and  25  different  samples  of  cow  dung,  includ- 
ing about  3500  different  colonies  isolated  by  various 
methods,  only  about  5  per  cent  belonged  to  the  class 
fermenting  dextrose  but  not  lactose.  In  rivers  and 
ground  waters,  on  the  other  hand,  this  group  made 
up  24  per  cent  of  the  colon-like  organisms  present, 
and  in  lakes  with  long  storage,  58  per  cent.  The  table 
below  shows  the  relative  increase  of  the  lactose  negative 
forms  with  the  natural  purification  of  rivers  in  four 
dry  months  following  rain  and  also  their  relative 
increase  in  settled  and  filtered  water  as  compared  with 
the  raw  river  water.  The  numbers  dealt  with  are  too 
small  to  give  entirely  uniform  results,  but  the  general 
trend  is  clear. 

PERCENTAGE  OF  DEXTROSE-FERMENTING  ORGANISMS 
FAILING  TO  FERMENT  LACTOSE.  CALCUTTA  WATER 
SUPPLY 

(CLEMESHA,  1912*) 


Month         

Oct. 

Nov. 

Dec. 

Jan. 

Feb. 

Condition  of  river  

Heavy 

Very 

Clear- 

Clear 

Clear 

ram 

muddy 

ing 

Raw  river  water  (80  colonies) 

20 

27 

68 

48 

79 

Settled  water  (20  colonies)  .  . 

50 

16 

85 

7i 

99 

Filtered  water  (60  colonies)  . 

29 

33 

72 

98 

95 

THE  COLON  GROUP  OF  BACILLI  115 

Clemesha  confirmed  these  results  by  a  long  series 
of  examinations  of  naturally  and  artificially  polluted 
waters,  all  tending  to  show  that  with  fresh  pollution 
most  of  the  dextrose  fermenters  ferment  lactose  as 
well,  while  with  storage  there  is  a  relative  increase  in 
the  lactose-negative  forms.  Careful  studies  of  the 
history  of  faecal  mixtures  in  water  showed  that  the 
resistant  form  was  a  particular  type,  called  by  Clemesha 
Bacillus  P.  Houston  (1911),  reports  similar  results 
for  London  waters.  Of  12,744  specimens  of  raw  river 
water  containing  dextrose-fermenting  organisms  81 
per  cent  gave  positive  results  in  lactose  and  formed 
indol  as  well,  thus  indicating  the  presence  of  the  colon 
group.  Of  18,960  specimens  of  filtered  water  contain- 
ing dextrose-fermenting  organisms  only  51  per  cent 
gave  positive  results  in  lactose  and  formed  indol. 
Clemesha  (191 2b),  in  an  analysis  of  Houston's  results, 
shows  that  the  preponderance  of  dextrose-positive 
lactose-negative  forms  is  here  not  due  to  the  Bacillus  P, 
which  Clemesha  found  in  India,  but  to  two  different 
forms. 

From  all  these  investigations  it  is  clear  that  the  dex- 
trose broth  test  does  not  bear  a  constant  relation  to 
the  presence  of  the  colon  group,  since  another  type  of 
organisms  fermenting  dextrose  but  not  lactose  is  rela- 
tively much  more  abundant  in  stored  and  relatively 
pure  water,  particularly  in  warm  weather. 

Phelps  and  Hammond  (1909)  have  pointed  out  a 
rather  serious  error  in  the  routine  isolation  of  B.  coli, 
as  it  used  to  be  practised  in  this  country,  due  to  the 
presence  of  this  group  of  organisms  which  fail  to  form 


116       ELEMENTS  OF  WATER  BACTERIOLOGY 

gas  in  lactose  broth.  The  five  standard  tests  for  B. 
coli  which  have  been  most  generally  adopted  in  the 
United  States  included  gas  production  in  dextrose 
broth  and  coagulation  of  milk,  but  not  gas  production 
in  lactose  broth.  It  was  supposed  that  types  pro- 
ducing gas  and  acid  in  dextrose  media  and  coagulating 
milk,  but  failing  to  form  gas  in  lactose  broth,  would 
be  rare.  In  the  particular  polluted  well  studied  by 
Phelps  and  Hammond,  however,  such  forms  were 
very  common,  outnumbering  true  colon  bacilli  four 
to  one  during  the  latter  part  of  the  investigation  when 
the  pollution  was  less  recent.  Two  workers  following 
the  standard  methods  but  using  dextrose  broth  for 
enrichment  on  the  one  hand  and  lactose  broth  on  the 
other  would  in  50  per  cent  of  the  samples  tested  have 
reached  opposite  conclusions  as  to  the  presence  or  absence 
of  B.  coli,  the  isolations  begun  with  dextrose  broth 
being  apparently  positive  and  those  begun  with  lactose 
broth  being  negative. 

It  is  also  clear  on  the  other  hand  that  the  dextrose 
test  as  ordinarily  used  may  be  negative  when  colon 
bacilli  are  present.  This  is  due  to  the  interaction  of 
various  bacteria  in  the  fermentation  tube  and  to  the 
solution  and  escape  of  gases  which  often  prevents  the 
production  of  the  typical  gas  formula.  Of  43  cultures 
isolated  by  Fuller  and  Ferguson  (1905)  at  Indianapolis, 
1 8  showed  less  than  20  per  cent  of  gas  after  48  hours 
in  the  enrichment  tube,  and  n  showed  less  than  10 
per  cent.  Hale  and  Melia  (1910),  working  with  a  pure 
culture  of  B.  coli  in  unsterilized  water  (containing  no 
other  gas  former)  report  that  of  818  tubes  showing  gas 


THE  COLON  GROUP  OF  BACILLI  117 

only  474  or  58  per  cent  gave  between  25  and  70  per 
cent  of  gas  in  the  closed  arm  with  25-40  per  cent  carbon 
dioxide. 

Stamm  (1906)  and  others  have  pointed  out  that  the 
ratio  of  carbon  dioxide  to  hydrogen  changes  with 
the  age  of  the  culture.  At  first  the  proportion  of  the 
former  to  the  latter  is  as  two  to  one,  and  later,  in  the 
same  tube,  the  ratio  is  reversed.  More  recently, 
Longley  and  Baton  (1907),  in  one  of  the  ablest  and  most 
fruitful  of  recent  contributions  to  water  bacteriology, 
have  made  it  clear  that  neither  of  these  quantitative 
determinations  is  of  importance  if  made  in  an  ordinary 
open  tube.  They  show,  first,  that  the  total  amount 
of  gas  formed  by  B.  coli  varies  widely,  from  10  to  80 
per  cent,  the  mode  of  the  curve  being  found,  not  at  50, 
but  at  35  per  cent.  Secondly,  they  show  that  the 
proportion  of  carbon  dioxide  present  is  a  function  of 
the  total  amount  of  gas.  They  find  that  when  grown 
in  an  atmosphere  of  CCb,  B.  coli  produces  a  gas  which 
consists  of  about  3  parts  of  carbon  dioxide  to  one  of 
hydrogen.  Assuming  that  the  gas  originally  formed 
by  B.  coli  has  always  about  this  composition,  and  that 
the  absorption  of  CCb  by  the  medium  is  the  chief  cause 
of  the  differences  observed  in  the  gas  which  collects 
in  the  closed  arm,  the  gas  ratio  would  vary  directly 
with  the  amount  of  total  gas;  the  more  rapidly  gas  is 
formed,  the  greater  the  proportion  of  CO2  remaining 
unabsorbed.  Calculation  on  this  basis  gives  a  curve 
very  close  to  the  observed  data. 

These  criticisms  apply  only  to  the  fermentation 
test  made  in  an  open  tube  and  uncorrected  for  the 


118       ELEMENTS  OF  WATER  BACTERIOLOGY 

absorption  of  CCb.  Keyes  (1909)  and  others  have 
introduced  more  exact  methods  based  on  the  collec- 
tion and  analysis  of  all  gases  formed  in  a  vacuum 
and  in  a  paper  shortly  to  be  published  by  L.  A.  Rogers, 
W.  M.  Clark  and  B.  J.  Davis  of  the  Bureau  of  Animal 
Industry  (kindly  loaned  to  us  by  Mr.  Rogers)  it  is 
shown  that  the  gas  ratio  when  accurately  determined 
is  highly  characteristic  for  certain  members  of  the 
colon  group. 

Not  only  is  it  true  that  little  reliance  can  be  placed 
on  the  exact  gas  formula  in  the  open  dextrose  broth 
tube,  but  cultures  of  the  colon  group  may  be  actually 
overgrown  and  lost  by  the  multiplication  of  other 
forms.  This  is  particularly  true  when  the  waters  are 
heavily  polluted  or  when  large  samples  are  examined. 
Hunnewell  and  one  of  us  (Winslow  and  Hunnewell, 
i902b)  found  that  of  48  samples  of  certain  polluted 
river  waters  18  showed  B.  coli  when  i  c.c.  was  inoculated 
directly  into  dextrose  broth,  while  in  only  4  cases  was 
a  positive  result  obtained  after  preliminary  treatment 
of  100  c.c.  in  carbol  broth.  In  153  samples  from 
presumably  unpolluted  water  B.  coli  was  found  5 
times  in  i  c.c.  and  n  times  by  the  examination  of 
the  larger  sample.  It  wrill  be  noted  that  these  results 
were  obtained  with  carbol  broth  for  the  enrichment  of 
the  larger  samples  and  carbol  broth  is  less  liable  to 
overgrowths  than  dextrose  broth. 

Whipple  (Whipple,  1903)  notes  that  2.9  per  cent  of 
some  samples  of  water  examined  by  him  gave  positive 
tests  with  .1  c.c.  but  not  with  i  c.c.,  while  4.3  per 
cent  gave  positive  tests  with  .1  c.c.  or  i  c.c.  and  negative 


THE  COLON  GROUP  OF  BACILLI      119 

tests  with  10  c.c.  Again,  in  another  series  of  samples 
examined,  of  those  which  gave  positive  tests  in  smaller 
portions  5.3  per  cent  were  negative  in  10  c.c.,  4.7  per 
cent  in  100  c.c.,  and  7.7  per  cent  in  500  c.c. 

Fromme  (1910)  has  made  an  interesting  study  of 
this  point  and  reports  that  of  59  samples  of  water  of 
good  quality  which  showed  B.  coli  in  small  portions 
25  per  cent  gave  negative  results  in  larger  portions; 
while  of  654  samples  of  polluted  waters  33  per  cent 
gave  negative  results  in  large  portions  and  positive 
results  in  smaller  ones.  These  results  are  of  value  as 
indicating  the  greater  liability  to  loss  by  overgrowth 
in  polluted  waters;  but  the  absolute  figures  are  much 
higher  than  workers  in  this  country  obtain  when  the 
enrichment  cultures  are  carefully  watched  and  plat- 
ings made  from  them  at  an  early  period. 

The  Use  of  Phenol  Broth  as  an  Enrichment  Medium 
to  Check  Overgrowths.  As  has  already  been  stated, 
phenol  has  less  inhibitory  action  upon  B.  coli  than 
upon  normal  water-bacteria,  and  it  was  hoped  that  a 
broth  containing  this  substance  might  be  employed 
for  preliminary  enrichment  with  advantage,  its  inhibitory 
power  checking  the  overgrowing  forms,  but  not  B.  coli. 
This  medium  was  used  in  place  of  dextrose  broth  for 
many  of  the  studies  made  in  connection  with  the  Chicago 
drainage  canal  (Reynolds,  1902).  Phenol  broth  con- 
sists of  ordinary  broth  to  which  o.i  per  cent  phenol 
is  added,  and  the  method  of  procedure  is  to  add  i  c.c. 
of  the  water  to  10  c.c.  of  the  sterilized  phenol  broth 
and  incubate  at  body  temperature  for  24  hours.  Litmus- 
lactose-agar  plates  are  then  made  and  the  examination 


120        ELEMENTS  OF  WATER  BACTERIOLOGY 

of  the  red  colonies  carried  out  as  described  for  the 
dextrose-broth  method.  It  has  unfortunately  proved, 
however,  that  with  waters  of  fairly  good  quality  the 
phenol  interferes  with  the  colon  bacilli  themselves  to 
a  serious  extent.  The  dextrose  broth  furnishes  a  more 
delicate  test  than  the  carbol  broth  when  the  number  of 
colon  bacilli  present  is  small,  as  is  clearly  shown  by  the 
following  table  from  Irons: 

PROPORTION  OF  POSITIVE  RESULTS  IN  TESTS  OF  POL- 
LUTED AND  UNPOLLUTED  WATERS  BY  DEXTROSE 
FERMENTATION-TUBE  AND  CARBOL-BROTH  METHODS 

(IRONS,  1901) 


Dextrose 

Fermentation- 
tube. 

Carbol-broth 
Method. 

+       -        ? 

4            ? 

Polluted  waters  

33     3i 

5 

38    30      i 

Relatively  unpolluted  waters  

56     38     2 

5 

37     61     21 

The  Eijkman  Test.  Another  enrichment  test,  which 
has  been  extensively  used  in  Germany  for  checking 
the  development  of  overgrowing  forms  and  limiting 
the  bacteria  in  the  fermentation  tube  to  the  colon 
group,  is  the  Eijkman  test,  which  depends  on  the  use 
of  a  high  temperature  (46°)  (Eijkman,  1904).  There 
is  no  doubt  that  such  a  procedure  cuts  out  the  water 
bacteria,  and  Christian  (1905),  Neumann  (1906),  and 
Thomann  (1907)  have  reported  good  results  from  its 
use.  Hilgermann  (1909),  too,  urges  the  value  of  the 
Eijkman  test  and  concludes  that  the  colon-like  bacilli, 
which  fail  to  grow  at  46°,  are  characteristic  of  compara- 
tively unpolluted  waters.  Other  observers  maintain, 


THE  COLON  GROUP  OF  BACILLI  121 

and  apparently  with  good  reason,  that  the  conditions 
of  this  test  are  too  severe  and  eliminate  many  intestinal 
bacilli  of  undoubted  significance.  Nowack  (1907)  found 
that  laboratory  cultures  of  B.  coli  often  fail  to  produce 
gas  in  Eijkman's  medium  at  46°,  unless  large  numbers 
are  introduced.  With  some  strains  an  inoculation 
of  over  a  million  bacteria  was  necessary  to  cause  gas 
formation. 

Konrich  (1910)  compared  the  Eijkman  enrichment 
method  (dextrose  peptone  water  at  46°)  and  that  of 
MacConkey  (dextrose-bile-salt  peptone  water  at  42°) 
with  57  water  samples  and  obtained  only  about 
70  per  cent  as  many  positive  results  with  the  former 
as  with  the  latter  method.  With  artificial  emulsions 
of  pure  cultures  and  of  faeces  even  greater  differences 
were  manifest.  These  studies  showed  conclusively 
that  incubation  at  46°  prevents  the  development  of 
great  numbers  of  colon  bacilli  and  is  unsuitable  for  an 
enrichment  process.  In  comparing  the  two  media 
(dextrose  peptone  water  with  and  without  bile  salts) 
at  the  same  temperature,  37°,  he  obtained  essentially 
similar  results. 

Fromme  (1910)  has  shown  that  during  the  first  5 
hours  in  various  enrichment  media  colon  bacilli  multiply 
more  rapidly  at  46°  than  at  37°.  After  that  time, 
however,  their  development  is  checked.  At  12  hours 
the  numbers  at  the  two  temperatures  are  about  equal 
and  between  12  and  24  hours  the  numbers  increase 
much  more  rapidly  at  37°. 

The  Neutral  Red  Reaction.  Other  special  media 
have  been  suggested  for  rapid  routine  water  analysis, 


122       ELEMENTS  OF  WATER  BACTERIOLOGY 

of  which  those  containing  "  neutral  red,"  one  of  the 
safranine  dyes,  have  been  somewhat  fully  studied. 
Rothberger  (Rothberger,  1898)  first  pointed  out  that  B. 
coli  reduces  solutions  of  this  substance,  the  color  chang- 
ing to  canary-yellow  accompanied  by  green  fluorescence. 
Makgill  (Makgill,  1901),  Savage  (Savage,  1901),  and 
other  English  observers,  as  well  as  Braun  (1906),  in 
France,  report  favorable  results  from  the  use  of  this 
test;  but  according  to  American  standards,  Irons 
(Irons,  1902)  and  Gage  and  Phelps  (Gage  and  Phelps, 
1903)  conclude  that  the  group  of  organisms  giving  a 
positive  neutral  red  reaction  is  too  large  a  one  to  give 
very  valuable  sanitary  information. 

Stokes  (1904)  urged  the  use  of  lactose  broth  with 
the  addition  of  neutral  red,  and  believed  that  the  pro- 
duction in  this  medium  of  30-50  per  cent  of  gas  with  a 

-  gas  formula  and  the  change  of  neutral  red  to  canary 

yellow  in  the  closed  arm  of  the  fermentation  tube  was 
characteristic  for  B.  coli. 

The  Lactose  Bile  Test  for  the  Colon  Group.  On  the 
whole,  by  far  the  most  satisfactory  results  in  making  a 
rapid  test  for  the  colon  group  have  been  obtained  by 
the  use  of  media  containing  bile  salts,  a  procedure 
the  development  of  which  in  this  country  we  owe 
principally  to  Jackson  (1906). 

MacConkey  (1900)  long  ago  suggested  the  use  of 
media  containing  bile  salts  (sodium  taurocholate) 
for  the  differentiation  of  B.  coli  and  B.  typhi,  and  bile- 
salts  media  have  been  used  by  various  English  observers 
(MacConkey,  1901;  MacConkey  and  Hill,  1901)  for 


THE  COLON  GROUP  OF  BACILLI 


123 


the  isolation  of  sewage  bacteria.  Jackson  studied 
the  action  of  various  bile  media  and  showed  their 
selective  inhibitory  action  in  the  striking  table  quoted 
below.  His  important  contribution  to  the  subject, 
however,  was  the  discovery  that  ox  bile  itself  could 
be  used  as  a  culture  medium,  and  that  it  was  easier 
to  prepare,  cheaper  and  more  effective  than  combina- 
tions of  meat  infusion  with  the  purified  bile  salts. 

SELECTIVE  ACTION  OF  BILE  SALTS 

(JACKSON,  1906) 


Bacteria 

per  c.c. 

Uncon- 
taminated 
Well. 

Contami- 
nated Pond. 

Suspension 
of 
Faeces. 

Suspension 
of 
Faeces. 

Gelatin   20° 

O2O 

2700 

3  50  ooo 

900,000 

Agar   37° 

2? 

170 

450,000 

900,000 

Bile  agar,*  37°  

14 

43 

300,000 

900,000 

Lactose  bile  agar,*  37°  . 

0 

25 

250,000 

675,000 

Lactose  bile  agar,*  37°  . 

O 

17 

250,000 

6oo,OOO 

Bile  agar,  37°  

0 

16 

60,000 

900,000 

*  Bile  diluted,  i.i. 

Jackson  suggested  the  use  of  fresh  ox  bile  containing 
i  per  cent  of  lactose  as  a  presumptive  test  instead  of 
dextrose  broth.  In  particular  he  hoped  that  this  medium 
would  be  free  to  a  great  degree  from  the  negative  results 
due  to  overgrowths  in  polluted  waters.  He  reported 
275  examinations  of  badly  contaminated  waters,  in 
which  65  per  cent  of  the  samples  failed  to  give  the 
dextrose-presumptive  test,  and  only  10  per  cent  failed 
to  show  gas  in  lactose  bile.  In  a  more  recent  communica- 
tion, Jackson  (1907)  reports  that  in  the  examination  of 


124        ELEMENTS  OF  WATER  BACTERIOLOGY 


5000  samples  of  water  at  the  Mt.  Prospect  Laboratory, 
the  bile  medium  has  proved  uniformly  satisfactory. 
He  recommends  incubation  for  72  hours,  results  being 
commonly  obtained,  however,  after  48  hours;  and  he 
considers  any  tube  showing  25  per  cent  gas  as  positive. 
In  a  series  of  examinations  carried  out  at  the  Institute 
of  Technology,  16  per  cent  of  the  positive  tubes  showed 


COMPARATIVE    PRESUMPTIVE    TESTS    WITH    DEXTROSE 
BROTH  AND  LACTOSE  BILE 

(SAWIN,  1907) 


Source. 

Percentage  of  Samples  Giving  Positive  Tests  for 
B.  Coli. 

Dextrose  Broth. 

Lactose  Bile. 

O.I  C.C. 

I.OC.C. 

IO.O  C.C. 

O.I  C.C. 

I.OC.C. 

lo.oc.c. 

I     Deep  wells  

O 
0 
T5-° 

IO.O 
IO.O 

o 

IO.O 

o 

1.0 
IO.O 

15-7 

5-0 
10.5 

IO.O 

15-0 

o 

IO.O 

15-0 

21  .0 
4O.O 
26.0 

5-o 
35-o 

O 
O 

5-o 

5-2 
IO.O 
IO.O 

o 
o 

0 

o 

0 
5-2 

5-o 
iQ-5 

0 

5-o 

O 
6.0 
15.0 
31.0 
iS-o 
50.0 

i5-o 
30.0 

2     Shallow  wells 

3     Lake  
4     Lake  

5     Lake  
6     Lake  
7     Lake  
8     Lake  

Average,  Nos.  3,  4,  5, 
6,  7,  8  

8-3 
47.0 

26.3 
36.8 

II  .0 

72.2 

37-6 
55-i 

23.6 

55-5 

73-7 
68.9 

5-0 

50.0 
30.0 
40.0 

4-3 

75-0 
90.0 
73-5 

26.0 

84.2 
85.0 
78.1 

9     River  
10     River  
ii     River. 

Average.  Nos.  9,  10,  n 
1  2     Brook  

36.7 

47-7 
50.0 
25.0 

55-i 

63-2 
73-7 
25.0 

66.0 

72.2 
78.9 

8.2 

40.0 

60.0 

84.2 
87-5 

79-4 

90.0 
90.0 
93-7 

82.4 

84.2 
90.0 
81.2 

13     Drainage  

14     Sewage 

Average,  Nos.  12,  13,  14 

40.9 

53-9 

53-1 

77-2 

91.2 

85-1 

THE  COLON  GROUP  OF  BACILLI  125 

gas  in  24  hours,  73  per  cent  after  48  hours,  and  the 
remaining  27  per  cent  only  after  72  hours;  but  the 
Committee  on  Standard  Methods  (1912)  believes  that 
the  forms  which  fail  to  develop  in  48  hours  are  attenuated 
forms  of  little  practical  significance.  Sawin  (1907) 
reports  comparative  results  with  dextrose  broth  and 
bile  on  different  classes  of  waters,  the  most  striking 
of  which  are  tabulated  on  p.  124. 

Like  other  enrichment  methods  which  eliminate  com- 
peting forms  it  is  no  doubt  true  that  the  lactose  bile 
test  cuts  out  some  weak  colon  bacilli.  As  a  presumptive 
method,  however,  it  is  far  superior  to  dextrose  broth, 
giving  a  higher  proportion  of  positive  tests  with  polluted 
waters  and  a  lower  proportion  of  erroneous  positive 
tests  with  waters  of  good  quality.  In  an  examination 
of  176  surface  waters  in  eastern  Massachusetts,  carried 
out  under  our  direction,  B.  coli  was  isolated  70  times. 
The  dextrose-broth  test  was  positive  120  times,  an  error 
of  70  per  cent;  while  the  bile  test,  alone,  was  positive 
78  times,  an  error  of  only  n  per  cent.  The  tabulated 
results  of  these  experiments  indicates  fairly  the  merits 
of  the  bile  medium  for  preliminary  enrichment  and  as  a 
presumptive  test. 

PRELIMINARY  AND   COMPLETE  RESULTS  OF  DEXTROSE 
BROTH  AND  BILE  TESTS.     176  SURFACE-WATERS 


Preliminary  Positive 
Results. 
(Gas  Formation.) 

Final  Positive  Results. 
(B.  Coli). 

Dextrose  broth  

1  2O 

70 

Lactose  bile  

78 

6d 

126       ELEMENTS  OF  WATER  BACTERIOLOGY 


Hale  and  Melia  (1910)  have  also  made  a  valuable 
comparison  of  a  number  of  presumptive  tests  as  applied 
during  a  period  of  2  years  to  85  samples  of  Manhattan 
water  (surface)  and  160  samples  of  Brooklyn  water 
(largely  ground-water).  Their  principal  results  are 
tabulated  below. 

RESULTS  OF  VARIOUS  PRESUMPTIVE  TESTS 

(HALE  AND  MELIA,  1910) 
245  Samples  of  New  York  Water 


Medium. 

Percentage  of  Positive  Results  in 

O.I   C.C. 

I.O  C.C. 

10.0  c.c. 

Dextrose  broth  (standard  gas  formula)  .  .  . 
Lactose  bile 

0.4 
0.8 
0.8 

0.8 
6-3 

7.0 

8.2 

15-5 

12.0 
31-4 

30-7 
38.4 
51.2 

57-6 
73-5 

Lactose-peptone  bile  
Dextrose  broth,  all  tubes  showing  5%  gas 
transplanted  to  bile  

Dextrose  broth,  5%  gas  and  over,  called 
positive  

This  table  indicates  very  clearly  the  fallacies  of  the 
dextrose  broth  tube.  Counting  all  gas  formers  in  this 
medium  as  positive  indicates  much  too  high  a  value 
and  including  only  the  tubes  showing  the  standard  gas 
formula  gives  much  too  low  a  value.  This  conclusion 
is  based  on  the  assumption,  warranted  by  the  results 
of  many  workers,  that  incubation  in  dextrose  broth 
followed  by  reinoculation  into  lactose  bile  (fourth  line 
of  the  table)  gives  with  reasonable  accuracy  the  real 
number  of  colon  bacilli  present.  By  this  standard 
the  use  of  plain  lactose  bile  with  these  waters  is  seen 
to  give  results  which  are  also  much  too  low;  but  lac- 
tose peptone  bile  approximates  closely  to  the  truth. 


.   THE  COLON  GROUP  OF  BACILLI      127 

Of  course  it  must  be  remembered  that  the  advantages 
of  lactose-bile  over  dextrose  broth  are  partly  due  to 
the  inhibiting  effect  of  the  bile  salts  and  partly  to  the 
use  of  lactose  instead  of  dextrose  which  cuts  out  the 
dextrose-positive  lactose-negative  group  to  which  allusion 
has  been  made  earlier  in  the  chapter.  The  relative 
importance  of  these  two  factors,  lactose  and  bile,  is 
well  brought  out  in  a  study  by  Stokes  and  S toner  (1909). 
These  authors  have  compared  a  considerable  series 
of  preliminary  enrichment  tests  followed  by  final 
isolation  in  dextrose  broth,  lactose  broth  and  lactose 
bile.  Of  567  colonies  from  positive  dextrose  broth 
tubes  only  52  per  cent  were  colon  bacilli;  of  3752 
colonies  from  positive  lactose  broth  and  lactose  bile 
tubes,  88  per  cent  of  the  lactose  broth  colonies  and  95 
per  cent  of  the  lactose  bile  colonies  were  B.  coli. 

With  sewages  and  heavily  polluted  waters  in  par- 
ticular the  lactose-bile  medium  has  proved  of  the 
greatest  value.  When  a  large  proportion  of  sewage 
is  present  the  colon  bacilli  are  fresh  from  the  intestine 
and  apparently  able  to  resist  the  antiseptic  salts.  On 
the  other  hand,  the  high  numbers  of  other  bacteria 
present  make  the  danger  of  overgrowths  particularly 
great.  With  waters  of  fair  quality,  such  as  those 
with  which  we  ordinarily  deal  in  sanitary  water  analysis, 
lactose  bile  is  open  to  the  same  objection  as  phenol 
broth  and  the  Eijkman  test  though  in  less  degree. 
It  inhibits  not  only  the  overgrowing  forms  but  the 
weaker  representatives  of  the  B.  coli  group  itself,  and 
the  net  effect  is  to  diminish  positive  results. 

Hale  and  Melia  (1910)  inoculated  unsterilized  water 


128       ELEMENTS  OF  WATER  BACTERIOLOGY 

(shown  to  contain  no  gas  formers)  with  a  pure  culture 
of  B.  coli  and  stored  it  for  different  periods  and  under 
different  conditions,  testing  at  intervals  by  various 
presumptive  tests.  The  colon  bacilli  lived  for  8-10 
days  at  37°,  for  38-75  days  at  20°,  and  77-84  days 
at  8°  C.  Comparison  of  presumptive  tests  with  plate 
counts  on  litmus-lactose  agar  showed  that  a  gas  test 
in  dextrose  broth  corresponded  to  an  average  of  4 
bacteria  and  a  positive  test  in  lactose  bile  to  39  bacteria. 
In  general  the  dextrose  broth  showed  gas  in  one  dilu- 
tion higher  than  the  lactose  bile;  and  the  difference 
increased  with  the  attenuation  due  to  prolonged  sojourn 
in  water. 

These  results  of  Hale  and  Melia  make  it  clear  that 
the  selective  action  of  bile  salts  upon  the  various  mem- 
bers of  the  colon  group  may  perhaps  be  an  advantage 
rather  than  a  disadvantage  for  practical  sanitary 
purposes.  The  Committee  on  Standard  Methods  (1912) 
recognizes  that  lactose  bile  does  inhibit  certain  weaker 
members  of  the  colon  group,  but  believes  that  these 
attenuated  organisms  indicate  only  remote  pollution 
and  are  of  little  significance.  They  say,  "  In  the 
interpretation  of  the  sanitary  quality  of  a  water, 
it.  is  best  to  discount  the  presence  of  attenuated 
B.  coli  and  to  be  sure  to  obtain  all  vigorous  types. 
The  lactose  bile  medium  accomplishes  both  of  these 
objects." 

The  advantages  of  the  dextrose  broth  enrichment  of 
weak  colon  bacilli  and  of  the  elimination  of  gas-forming 
organisms  other  than  B.  coli  by  bile  may  both  be  obtained 
as  pointed  out  above  by  inoculating  lactose  bile  from 


THE  COLON  GROUP  OF  BACILLI        .      129 

the  dextrose  broth.  Hale  and  Melia  (1910)  find  that 
this  gives  results  in  as  high  a  dilution  as  by  the  use  of 
dextrose  broth  and  with  the  clear-cut  results  of  lactose 
bile. 

The  Aesculin  Test.  A  test  for  the  colon  group  which 
has  attracted  much  interest  during  the  last  few  years 
is  the  fermentation  of  the  glucoside  aesculin.  B. 
coli  effects  a  hydrolytic  splitting  of  this  substance, 
producing  sugar  and  a  substance  called  aesculetin, 
which  reacts  with  iron  citrate  to  produce  a  dark  brown 
salt.  Harrison  and  van  der  Leek  (1909)  have  used 
broth  or  agar  made  up  with  i  or  2  per  cent  Witte's 
peptone,  .5  per  cent  sodium  taurocholate,  .1  per  cent 
aesculin  and  .05  per  cent  citrate,  and  find  that  the 
black  colonies  with  a  black  halo  produced  by  the  colon 
group  of  organisms  are  highly  characteristic.  Of 
60  samples  of  water  which  showed  blackening  of 
aesculin  broth,  all  proved  to  contain  B.  coli.  Hale 
and  Melia  (1911)  have  shown  that  two  species  of 
streptococci,  the  aurococcus,  and  the  bacillus  of 
pneumonia,  also  give  the  aesculin  reaction  and  that 
in  the  absence  of  the  bile  salt  which  the  aesculin 
medium  contains  a  number  of  other  forms  may  ferment 
this  glucoside. 

On  the  whole  we  do  not  think  it  has  been  shown 
that  aesculin  has  sufficient  differential  value  to  war- 
rant its  inclusion  in  enrichment  media  to  be  used  in 
the  colon  test.  It  appears  that  the  anaerobic  B.  welchii 
is  practically  the  only  form  outside  the  colon  group 
which  produces  a  characteristic  reaction  in  lactose 
bile  and  not  in  aesculin-bile  salt  media.  It  may  prove 


130       ELEMENTS  OF  WATER  BACTERIOLOGY 

worth  while  to  use  aesculin  to  exclude  this  form,  but 
its  occurrence  in  ordinary  water  work  is  so  rare  that  the 
extra  complication  seems  hardly  justified. 

The  Use  of  Synthetic  Media  for  the  Isolation  of  the 
Colon  Group.  Dolt  (1908),  working  in  Prof.  Gorham's 
laboratory  at  Brown  University,  has  attempted  with 
success  to  substitute  synthetic  media  of  simple  and 
known  composition  for  the  usual  meat-infusion-peptone 
media  used  in  the  isolation  of  B.  coli.  He  first  found 
that  colon  bacilli  will  grow  readily  on  a  medium  con- 
taining asparagin  and  sodium  or  ammonium  phosphate. 
He  then  attempted  to  substitute  for  asparagin  various 
simple  organic  substances  similar  in  their  structure 
to  the  cholic  acid  of  the  bile  which  exerts  a  selective 
action  in  favor  of  the  colon  group.  He  finally  succeeded 
in  preparing  two  media  which  promise  to  be  of  con- 
siderable value  in  permitting  the  growth  of  B.  coli 
while  checking  other  forms.  The  first  of  these  media 
is  made  up  as  follows:  500  c.c.  of  a  3  per  cent  solution 
of  purified  agar  is  mixed  with  an  equal  portion  of  a 
solution  of  i  per  cent  glycerin  and  0.2  per  cent 
(NH^HPCU.  It  is  neutralized  with  sodium  hydroxide 
and  i  per  cent  of  lactose  is  added  before  sterilization. 
In  the  second  medium  5  gm.  of  ammonium  lactate  is 
substituted  for  the  glycerin  and  i  gm.  of  Na2HPO4 
for  the  ammonium  phosphate.  These  media  proved 
to  have  a  considerable  selective  value,  cutting  out  most 
of  the  water  bacteria;  but  like  all  such  selective  media 
they  cut  out  a  good  many  of  the  colon  bacilli  too.  The 
results  of  a  single  test  are  shown  in  the  table  below. 
The  procedure  well  merits  further  study,  however. 


THE  COLON  GROUP  OF  BACILLI 


131 


COMPARATIVE  RESULTS  OF  ORDINARY  AND  SYNTHETIC 
AGAR  MEDIA 

(DOLT,   1908) 


Total  Colonies. 

Red  Colonies. 

B.  Coli. 

Standard  agar  

67 

38 

18 

Glycerin  agar  

27 

9 

9 

Ammonium  lactate  agar 

I? 

9 

9 

The  Use  of  Liver  Broth  for  the  Isolation  of  a  Maximum 
Proportion  of  Gas-forming  Bacteria.  The  media  we 
have  been  discussing,  phenol  broth,  dextrose  broth 
incubated  at  46°,  and  bile,  are  designed  to  cut  down 
the  gas  producers  which  appear  in  ordinary  dextrose 
broth  so  that  only  vigorous  typical  members  of  the 
colon  group  are  able  to  develop.  For  special  research 
purposes  when  it  is  desired  to  get  the  largest  possible 
proportion  of  gas  formers  of  all  kinds,  there  are  other 
media  which  give  even  more  positive  fermentation 
results  than  dextrose  broth  itself.  The  most  important 
of  these  is  the  liver  broth  of  Jackson  and  Muer  (1911) 
made  up  with  beef  liver,  peptone,  dextrose  and  potas- 
sium-acid-phosphate. The  Committee  on  Standard 
Methods  of  Water  Analysis  (1912)  recommends  that 
if  "  a  study  of  all  gas-forming  bacteria,  including 
attenuated  forms,  is  desirable,  then  liver  broth  should 
be  employed  in  preference  to  the  usual  dextrose  broth, 
as  it  gives  a  larger  number  of  attenuated  forms,  has 
better  rejuvenating  power,  and  gives  fewer  anomalies 
and  greater  and  more  rapid  gas  production."  In 
order  to  avoid  attenuation  or  inhibition  transplants 


132       ELEMENTS  OF  WATER  BACTERIOLOGY 

should  be  made  from  this  enrichment  medium  after 
6-12  hours  at  37°. 

Isolation  of  Pure  Cultures  from  the  Enrichment  Tube. 
In  case  one  does  not  rely  upon  a  "  presumptive  "  test 
alone  but  desires  to  study  the  organisms  present  in 
detail  the  isolation  upon  a  solid  medium,  usually 
litmus-lactose-agar  in  this  country,  must  follow  the 
enrichment  process.  Since  the  enrichment  tube  was 
inoculated  with  a  known  amount  of  water  all  further 
work  is  purely  qualitative,  and  it  is  only  necessary  to 
obtain  such  a  number  of  colonies  upon  the  lactose  plate 
that  the  isolation  of  a  pure  culture  shall  be  easy.  In 
practice  the  following  procedure  has  been  found  gen- 
erally successful.  After  the  enrichment  tubes  have 
been  incubated  for  12  to  24  hours  at  37°,  from  those 
which  show  gas,  one  loopful  is  carried  over  to  a  tube 
containing  10  c.c.  of  sterile  water,  and  of  this  water  one 
loopful  is  taken  for  the  inoculation  of  the  plate. 
Ordinarily  this  will  give  colonies  which  are  sufficiently 
well  separated,  but  a  second  plate,  inoculated  from  the 
dilution  water  with  a  straight  needle  instead  of  a  loop, 
furnishes  a  desirable  safeguard.  With  practice  it  is 
possible  to  effect  a  proper  seeding  more  rapidly  by 
barely  touching  the  tip  of  a  straight  needle  to  the  broth 
in  the  fermentation  tube  and  transferring  this  directly 
to  the  agar.  The  touch  must  be  a  very  light  one,  how- 
ever, or  the  colonies  on  the  plate  will  be  too  thick  for 
proper  isolation. 

The  litmus-lactose-agar  plates  made  in  this  manner 
should  be  incubated  for  from  12  to  24  hours  at  the  body 
temperature  (37°),  at  the  end  of  which  time,  if  B.  coli  is 


THE  COLON  GROUP  OF  BACILLI  133 

present,  red  colonies  upon  a  blue  field  will  be  visible. 
The  Htmus-lactose-agar  plate  may  become  blue  again 
after  48  hours,  owing,  presumably,  to  the  formation  of 
amines  and  ammonia  by  the  action  of  the  bacteria 
upon  the  nitrogenous  matter  present.  If  the  dilution 
is  too  low,  the  resulting  colonies  will  be  small  and 
imperfectly  developed,  making  it  difficult  to  be  sure 
of  pure  cultures  for  the  subsequent  tests.  A  great 
number  of  colonies  will  also  prevent  the  change  of 
reaction  from  acid  back  to  alkaline. 

In  the  selection  of  those  red  colonies  which  are  to  be 
fished  from  the  litmus-lactose-agar  plate  the  appearance 
of  the  growths  must  be  closely  noted.  A  colony  of 
irregular  contour,  surrounded  by  a  very  faint  area  of 
reddening,  will  probably  belong  to  some  member  of  the 
B.  mycoides  group  (Winslow  and  Nibecker,  1903); 
small,  compact,  bright-red  colonies  are  characteristic 
of  the  streptococci,  and  Gage  and  Phelps  (Gage  and 
Phelps,  1903)  have  pointed  out  that  of  these  there  are 
two  types,  one  of  a  brick-red  color,  and  of  such  con- 
sistency as  to  be  readily  picked  up  by  the  needle-point, 
and  the  other  smaller  and  of  an  intense  vermilion 
color.  The  colonies  of  the  colon  bacillus  are  usually 
well  formed,  pulvinate  on  the  surface  and  fusiform 
when  growing  deeper  down. 

If  no  red  colonies  appear  on  the  litmus-lactose-agar 
plate  after  a  positive  result  in  dextrose  broth  one  of 
four  things  has  occurred:  There  may  be  an  organism 
present  which  forms  gas  in  dextrose  but  no  acid  in 
lactose;  there  may  be  present  forms  which  individually 
fail  to  attack  lactose  but  growing  together,  symbiotically, 


134       ELEMENTS  OF  WATER  BACTERIOLOGY 

produce  gas  in  dextrose;  B.  welchii  or  some  other 
form  which  will  not  grow  on  aerobic  plates  may  have 
produced  the  gas;  or  an  organism  orginally  present 
and  capable  of  fermenting  both  sugars  may  have  been 
overgrown  and  lost  in  the  enrichment  tube.  If  plates 
are  made  on  the  first  appearance  of  gas  the  likelihood 
of  the  latter  possibility  will  be  reduced  to  a  minimum. 
Neither  of  the  first  two  contingencies  has  any  sanitary 
significance;  as  we  have  seen,  bacteria  which  ferment 
dextrose  and  not  lactose  are  not  specially  characteristic 
of  pollution.  In  general,  therefore,  the  absence  of  red 
colonies  on  the  agar  plate  may  be  considered  a  negative 
result.  If  red  colonies  are  present  they  must  be  sub- 
cultured  and  examined  further. 

The  agar  streak  made  from  the  litmus-lactose-agar 
plate  shows  after  24  hours  certain  marked  character- 
istics. The  most  distinct  types  are  two,  the  abundant, 
first  translucent,  later  whitish  and  cheesy  growth, 
covering  nearly  the  whole  surface  of  the  agar,  character- 
istic of  B.  coli  and  its  allies,  and  a  very  faint  growth, 
either  confined  strictly  to  the  streak  or  made  up  of  faint 
isolated  colonies,  dotted  here  and  there  over  the  surface. 
The  latter  cultures  are  typical  of  the  sewage  streptococci, 
and  a  microscopic  examination  will  generally  settle 
their  status  at  once.  Of  the  more  luxuriant  growths, 
some  of  which  are  stringy  to  the  needle,  many  will 
generally  prove  to  be  atypical,  and  if  any  of  the  weakly 
fermenting  forms  (B.  mycoides)  are  present  a  dull 
wrinkled  growth  will  be  produced. 

The  various  tests  which  may  be  applied  to  the  cul- 
tures after  they  have  been  isolated,  the  subgroups 


THE  COLON  GROUP  OF  BACILLI  135 

into  which  the  colon  group  may  be  divided  by  their 
use,  and  the  significance  of  the  results  obtained  will 
be  discussed  in  Chapter  VIII. 
Practical  Routine  Test  for  the  Colon  Group.     As  has 

been  pointed  out  above  the  aggregation  of  lactose- 
fermenting  bacilli  which  we  call  the  colon  group  may 
be  almost  indefinitely  subdivided  by  the  application 
of  a  more  or  less  elaborate  series  of  diagnostic  tests. 
Each  observer  in  the  past  drew  up  a  scheme  of  what 
he  believed  to  be  essential  tests  and  called  all  the 
bacteria  which  failed  to  conform  to  them  "  atypical." 
The  more  of  such  "  atypical  "  forms  a  particular 
worker  includes  the  greater  will  be  the  number  of  positive 
isolations.  The  definition  of  this  or  any  other  bacterial 
species  is  more  or  less  arbitrary;  we  consider  as  true 
colon  bacilli  those  which  fulfil  a  particular  set  of  tests, 
and  class  as  pseudo-colon  organisms  those  which  do 
not.  If  we  find,  having  established  such  an  arbitrary 
standard,  that  the  colon  bacillus,  as  determined  by  it, 
is  found  in  waters  known  to  be  polluted,  and  not,  as 
a  rule,  in  those  known  to  be  free  from  pollution,  the 
sanitarian  can  afford  to  ignore  the  theoretical  question 
of  specific  values  and  make  confident  use  of  the  practical 
test.  In  order  that  results  may  rest  on  a  sound  basis 
of  comparable  data  for  various  waters,  it  is  of  course 
essential,  however,  that  a  standard  set  of  reactions 
should  be  agreed  upon  by  sanitary  bacteriologists. 

After  a  considerable  period  of  uncertainty,  in  which 
each  observer  used  the  procedure  which  happened  to 
appeal  to  him,  the  attainment  of  comparative  results 
was  made  possible  by  the  establishment  of  standard 


136       ELEMENTS  OF  WATER  BACTERIOLOGY 

methods  of  procedure  by  bodies  of  authoritative  posi- 
tion, both  in  England  and  America.  In  1904  an 
English  Committee,  appointed  to  consider  the  Stand- 
ardization of  Methods  for  the  Bacterioscopic  Examina- 
tion of  Water,  presented  a  series  of  obligatory  tests 
and  optional  tests;  and  in  1905  the  Committee  on 
Standard  Methods  of  Water  Analysis  of  the  American 
Public  Health  Association  drew  up  a  set  of  diagnostic 
characters  for  B.  colL  The  latter  corresponded  in 
general  with  the  plan  developed  by  the  Massachusetts 
State  Board  of  Health  (Massachusetts  State  Board  of 
Health,  1899)  and  long  in  use  at  the  Massachusetts 
Institute  of  Technology,  and  involved  the  determina- 
tion of  morphology,  motility,  fermentation  of  dex- 
trose broth,  coagulation  of  milk,  production  of  indol 
and  reduction  of  nitrates.  The  English  standard  pro- 
cedure corresponded  quite  closely  to  this  (Committee 
appointed  to  consider  the  Standardization  of  Methods 
for  the  Bacterioscopic  Examination  of  Water,  1904), 
although  it  differed  from  the  American  method  in  cer- 
tain respects. 

Since  these  standards  were  formulated  8  years  ago 
their  artificial  nature  has  been  made  more  and  more 
manifest  to  those  who  have  used  them.  A  still  more 
fundamental  question,  however,  has  pressed  itself 
upon  the  practical  analyst.  Each  lactose  bile  presump- 
tive test  involves  the  use  of  a  single  tube  of  medium 
and  the  work  is  complete  in  48  hours.  The  "  com- 
plete "  test  as  used  in  America  required  the  use  of 
seven  different  media  and  took  9  days  to  complete, 
since  the  gelatin  subculture  must  be  incubated  for 


THE  COLON  GEOUP  OF  BACILLI      137 

at  least  a  week.  Granting  that  the  lactose  bile  test 
gave  us  the  whole  colon  group  and  that  the  "  typical 
B.  coli  "  giving  characteristic  reactions  in  milk,  peptone, 
gelatin  and  the  nitrate  solution,  only  the  more  sensitive 
members  of  the  group,  indicative  of  recent  pollution, 
was  the  extra  information  gained  worth  the  additional 
trouble?  Under  the  conditions  which  generally  obtain, 
in  the  United  States  at  least,  it  appears  not.  The  colon 
test  at  best  is  an  approximate  one,  and  its  results 
are  usually  only  expressed  in  decimal  fractions,  positive 
in  10  c.c.,  i  c.c.,  or  .1  c.c.  for  example.  From  70  to 
90  per  cent  of  the  bacteria  which  give  the  lactose  bile 
test  prove  to  be  "  typical  "  B.  coli  on  any  of  the  defini- 
tions ordinarily  used.  This  makes  a  difference  so  slight 
as  to  be  almost  negligible.  We  cannot  condemn  a 
water  because  it  contains  10  rather  than  7  colon  bacilli 
in  a  given  proportion.  It  may  be  that  under  tropical 
conditions  such  as  those  described  by  Clemesha  (to 
be  discussed  in  a  following  chapter)  certain  forms  of 
the  colon  group  persist  for  a  long  time  in  stored  waters 
from  which  disease  germs  have  disappeared.  These 
resistant  forms  must,  however,  be  studied  by  a  much 
more  elaborate  procedure  than  the  7  tests  in  the  old 
American  Standard  method;  and  it  seems  clear  that 
they  do  not  occur  in  large  numbers  in  temperate  cli- 
mates. For  our  conditions  the  whole  group  of  forms 
which  produce  gas  in  lactose  bile  should  be  absent  from 
safe  waters. 

The  Committee  on  Standard  Methods  of  Water 
Analysis  in  its  last  report  (1912)  apparently  takes 
this  ground,  although  its  discussion  of  the  problem 


138       ELEMENTS  OF  WATER  BACTERIOLOGY 

is  distinctly  ambiguous.  In  one  section  of  the  report 
"  Recommended  Procedures  for  Treating  Samples  " 
complete  isolation  and  the  use  of  the  old  confirmatory 
tests  in  fermentation  tubes,  milk,  gelatin  tube,  peptone 
solution  and  nitrate  broth  are  discussed.  In  another 
place  it  is  pointed  out  that  the  entire  colon  group  is 
typical  of  the  presence  of  faecal  matter  and  the  follow- 
ing "  Quantitative  Test  for  the  B.  coli  Group  "  is 
recommended : 

"  Add  the  quantities  of  water  or  sewage  to  be  tested 
in  dilutions  by  tenths,  sufficient  in  number  to  obtain 
a  negative  test,  to  fermentation  tubes  holding  at  least 
40  c.c.  of  lactose  bile,  incubate  at  37°  C.  and  note 
the  production  of  gas.  Gas  often  forms  in  a  few  hours 
when  large  numbers  of  B.  coli  are  present,  but  the 
standard  time  for  observing  gas  production  is  48  hours. 
Small  numbers  of  somewhat  attenuated  B.  coli  may 
require  3  days  to  form  gas.  Attenuated  B.  coli  does 
not  represent  recent  contamination  and  all  B.  coli 
not  attenuated  grows  readily  in  lactose  bile.  No 
other  organism  except  B.  welchii  gives  such  a  test  in 
lactose  bile.  B.  welchii  is  of  rather  rare  occurrence 
in  water,  is  of  faecal  origin,  is  almost  invariably  accom- 
panied by  B.  coli,  and  while  the  sanitary  significance 
is  the  same  it  may  if  desired  be  distinguished  from  B. 
coli  by  a  microscopical  examination  of  the  bile  solution 
when  long  strings  of  much  larger  bacilli  than  B.  coli 
are  seen." 

So  far  as  we  can  judge  from  the  report  this  appears 
to  constitute  the  preferred  procedure  of  the  Committee. 
In  any  case  the  matter  was  passed  upon  by  the  Labora- 


THE  COLON  GROUP  OF  CACILLI  139 

tory  Section  of  the  American  Public  Health  Associa- 
tion at  its  Washington  meeting  in  September,  1912, 
by  the  adoption  of  a  resolution  recommending  deter- 
minations of  numbers  at  20°  and  37°  and  the  lactose 
bile  presumptive  test  as  the  standard  routine  procedure 
in  water  examinations.  The  detailed  study  of  particular 
types  of  the  colon  group  may,  of  course,  be  important, 
in  special  cases;  but  the  lactose  bile  test  is  sufficient 
for  general  sanitary  purposes. 


CHAPTER  VII 

SIGNIFICANCE  OF  THE  PRESENCE  OF  THE  COLON  GROUP 
IN  WATER 

Colon  Bacilli  in  the  Intestines  of  the  Lower  Animals. 

The  Bacillus  coli  i »  by  no  means  confined  to  the  human 
intestine.  Dyar  and  Keith  (Dyar  and  Keith,  1893)  found 
it  to  be  the  prevailing  intestinal  form  in  the  cat,  dog,  hog, 
and  cow.  About  the  same  time,  Fremlin  (Fremlin,  1893) 
found  colon  bacilli  in  the  faeces  of  dogs,  mice,  and  rab- 
bits, but  not  in  those  of  rats,  guinea  pigs,  and  pigeons. 
Smith  (Smith,  1895)  recorded  the  presence  of  the 
organism,  in  almost  pure  cultures,  in  the  intestines  of 
dogs,  cats,  swine,  and  cattle;  and  he  also  found  it  in 
the  organs  of  fowls  and  turkeys  after  death.  Brotzu 
(Brotzu,  1895)  reported  B.  coli  and  allied  forms  as  very 
abundant  in  the  intestine  of  the  dog;  and  Belitzer 
(Belitzer,  1899)  isolated  typical  colon  bacilli  from  the 
intestinal  contents  of  horses,  cattle,  swine,  and  goats. 
Moore  and  Wright  (Moore  and  Wright,  1900)  recorded 
the  finding  of  the  colon  bacillus  in  the  horse,  cow, 
dog,  sheep,  and  hen;  and  in  a  later  report  (Moore 
and  Wright,  1902)  they  noted  its  occurrence  in  swine  and 
in  some,  but  not  all,  the  specimens  of  rabbits  examined. 
In  frogs  it  was  not  found.  Eyre  (1904)  has  more 
recently  isolated  typical  B.  coli  from  the  intestines 

140 


SIGNIFICANCE  OF  COLON  GROUP  IN  WATER     141 

of  mice,  rats,  guinea  pigs,  rabbits,  cats,  dogs,  sheep, 
goats,  horses,  cows,  hens,  ducks,  pigeons,  sparrows, 
divers,  gulls,  and  fish  of  various  sorts.  Houston  (1904) 
found  B.  coli  abundant  in  the  faeces  of  gulls,  as  might  be 
expected  from  their  feeding  habits.  Houston  (1905) 
and  other  recent  observers  have  found  it  impossible, 
even  by  the  use  of  elaborate  series  of  fermentation  tests, 
to  distinguish  human  B.  coli  from  those  found  in  animals. 
Savage  (1906)  compared  colon-like  organisms  isolated 
from  the  intestines  of  swine,  cattle,  horses,  and  sheep 
with  those  of  human  origin  in  respect  to  their  action 
upon  lactose,  dulcite,  mannite,  raffinose,  glycerine, 
maltose,  galactose,  lamilose,  saccharose,  starch  and 
cellulose;  but  he  failed  to  find  any  general  correlations 
between  habitat  and  biochemical  powers. 

Ferreira,  Horta  and  Paredes  (i9o8b)  have  made 
perhaps  the  most  elaborate  study  of  the  distribution 
of  colon  bacilli  in  the  lower  animals.  They  isolated 
8 1  lactose-fermenting  bacilli  from  38  species  of  mammals 
and  8  species  of  birds,  including  monkeys,  bears,  wolves, 
foxes,  hyenas,  lions,  panthers,  tapirs,  a  camel,  deer, 
and  ostriches  from  the  Zoological  Gardens.  These 
cultures  were  studied  by  an  elaborate  series  of  tests 
and  93  per  cent  of  them  proved  to  be  typical  B.  coli. 
Bettencourt  and  Borges  (i9o8b)  working  in  the  same 
laboratory  showed  that  there  were  no  specific  differences 
in  agglutination  with  immune  sera  and  in  complement 
fixation  between  the  colon  bacilli  of  human  and  of 
animal  origin.  Konrich  (1910)  reports  the  examina- 
tion of  170  samples  of  faeces  from  men,  horses,  swine, 
sheep,  cows,  goats,  dogs,  cats,  guinea  pigs,  mice,  rabbits, 


142       ELEMENTS  OF  WATER  BACTERIOLOGY 

rats,  earthworms,  moles,  fowls,  swallows,  sparrows, 
ducks,  pigeons,  geese,  a  jackdaw,  a  redstart,  a  blackbird, 
an  adder,  and  a  trout.  Three  out  of  5  guinea  pig 
samples,  4  out  of  20  horse  samples,  2  out  of  3  mouse 
samples,  3  out  of  8  rabbit  samples,  and  2  out  of  8 
earthworm  samples,  14  in  all,  were  negative;  while 
all  the  rest  showed  B.  coli. 

In  cold-blooded  animals  the  occurrence  of  B.  coli  is 
less  constant.  Negative  results  in  the  frog  and  positive 
results  in  certain  fishes,  an  adder  and  earthworms  have 
just  been  quoted.  Amyot  (1902)  failed  to  find  the 
organism  in  the  intestines  of  23  fish  representing  14 
species.  Johnson,  on  the  other  hand  (Johnson,  1904), 
in  the  examination  of  the  stomach  and  intestines  of 
67  fish  caught  in  the  polluted  Illinois  and  Mississippi 
Rivers,  isolated  B.  coli  47  times.  He  concluded  from 
these  results  that  the  migration  of  fish  from  a  con- 
taminated stream  or  lake  to  an  unpolluted  one  may 
explain  the  occasional  finding  of  B.  coli  in  small  samples, 
or  the  more  regular  detection  of  it  in  large  volumes 
of  the  water. 

Bettencourt  and  Borges  (i9o8b)  isolated  29  cultures 
of  colon-like  microbes  from  the  intestines  of  17  types 
of  fishes,  reptiles  and  amphibia.  Only  8  of  the  29 
formed  gas  in  lactose  broth  and  only  2  (from  an  eel 
and  an  adder)  proved  to  be  typical  B.  coli.  It  should 
be  noted,  however,  that  the  samples  of  faecal  material 
were  plated  directly  on  Endo  medium  instead  of  being 
subjected  to  the  more  sensitive  process  of  preliminary 
enrichment. 

Fromme  (1910)  reviews  the  work  of  many  observers 


SIGNIFICANCE  OF  COLON  GROUP  IN  WATER     143 

in  regard  to  the  presence  of  colon  bacilli  in  the  intes- 
tines of  cold-blooded  animals  (particularly  fish  of 
various  sorts  and  oysters)  and  concludes  that  while 
they  are  regularly  found  in  warm-blooded  animals 
they  are  found  often,  but  not  regularly,  in  cold-blooded 
animals.  The  lower  the  zoological  type  the  rarer  are 
the  colon  bacilli. 

Alleged  Ubiquity  of  the  Colon  Bacillus.  Many  bacte- 
riologists have  gone  further  and  affirmed  that  the 
colon  bacillus  was  not  a  form  characteristic  of  the 
intestine  at  all,  but  a  saprophyte  having  a  wide  dis- 
tribution in  nature.  The  first  of  this  school,  perhaps, 
was  Kruse  (Kruse,  1894),  who  in  1894  protested  against 
the  arbitrary  conclusions  drawn  from  the  colon  test 
as  then  applied.  He  pointed  out  that  the  characters 
usually  observed  marked,  not  a  single  species,  but  a 
large  group  of  organisms.  As  ordinarily  defined,  he 
added,  "  the  Bacterium  coli  is  in  no  way  characteristic 
of  the  faeces  of  men  or  animals.  Such  bacteria  occur 
everywhere,  in  air,  in  earth,  and  in  the  water,  from  the 
most  different  sources."  Even  if  the  relations  to  milk 
and  sugar  media  be  considered,  "  micro-organisms 
with  these  characteristics  are  also  widespread."  Dr. 
Kruse  gave  no  experimental  data  on  which  his  opinion 
was  based.  In  the  same  year  Beckmann  (Beckmann, 
1894)  isolated  a  bacillus  which  he  identified  by  pretty 
thorough  tests  as  B.  coli  from  the  city  water  of  S trass- 
burg,  a  ground-water  which  he  believed  could  by  no 
possibility  be  subject  to  faecal  contamination.  Large 
quantities  of  water  were  used  for  the  isolation. 

Refik  (Refik,   1896)  recorded  the  constant  presence 


144       ELEMENTS  OF  WATER  BACTERIOLOGY 

of  colon  bacilli  in  water  of  all  sorts,  public  supplies, 
wells,  cisterns,  and  springs  in  the  neighborhood  of 
Constantinople,  and  Poujol  in  the  succeeding  year 
reported  (Poujol,  1897)  the  isolation  of  B.  coli  from 
22  out  of  34  waters  studied  by  him  in  relation  to  their 
use  as  public  supplies.  The  waters  were  from  various 
sources — springs,  wells,  and  rivers — but  all  were  of  fair 
quality  and  many  quite  free  from  any  possibility  of 
contamination.  Samples  of  100  c.c.  were  used  for 
analysis. 

Certain  Italian  observers  appear  to  have  come  to 
even  less  conservative  conclusions.  Abba  (Abba,  1895) 
found  colon  bacilli  constantly  present  in  unpolluted 
waters  near  Turin.  Moroni  (Moroni,  1898;  Moroni, 
1899)  reported  the  examination  of  numerous  deep  and 
shallow  wells  and  unpolluted  springs  about  Parma, 
as  well  as  of  the  public  water-supply  of  the  city,  and 
concluded  that  the  colon  bacillus  was  a  water  form  and 
had  no  sanitary  significance.  The  characters  used 
for  the  identification  of  the  species  in  this  case  were 
fairly  exhaustive,  but  both  Abba  and  Moroni  used 
liter  samples  for  analysis. 

Levy  and  Bruns  (Levy  and  Bruns,  1899)  gave  a  new 
turn  to  the  discussion  by  emphasizing  the  importance  of 
animal  inoculation,  already  suggested  by  Blachstein 
(Blachstein,  1893)  and  others.  They  claimed  that  the 
existence  of  numerous  para-colon  and  para-typhoid 
organisms  in  air,  in  dust,  and  in  unpolluted  water 
made  it  impossible  to  decide  by  ordinary  bacteriological 
methods  whether  true  colon  bacilli  were  present  in 
water  or  not.  In  no  case,  however,  did  representatives 


SIGNIFICANCE  OF  COLON  GROUP  IN  WATER     145 

of  the  colon  group  isolated  by  them  from  water  kill 
a  guinea  pig,  even  when  i  or  2  c.c.  were  injected  intra- 
peritonally.  The  authors,  therefore,  considered  patho- 
genicity  as  an  attribute  belonging  only  to  the  true 
B.  coli  of  the  intestine.  This  paper  aroused  Professor 
Kruse's  pupil,  Weissenfeld,  to  a  publication,  in  which 
the  position  of  the  Bonn  school  was  carried  to  an 
extreme.  Weissenfeld  reported  (Weissenfeld,  1900)  the 
analysis  of  30  samples  of  water  supposedly  pure, 
and  of  26  samples  considered  to  be  contaminated.  In 
each  case  a  single  centimeter  sample  was  first  incubated 
in  Parietti  broth,  and  if  no  growth  occurred,  larger 
samples  of  half  a  liter  or  a  liter  were  examined.  Colon 
bacilli  were  found  in  all  the  samples;  and  the  patho- 
genicity  varied  independently  of  the  source  of  the  water. 
The  author  concluded  that  "  the  so-called  Bacterium 
coli  may  be  found  in  waters  from  any  source,  good  or 
bad,  if  only  a  sufficiently  large  quantity  of  the  water 
be  taken  for  analysis." 

With  regard  to  the  question  of  pathogenicity  as  a 
diagnostic  test  for  intestinal  B.  coli,  there  is  little  doubt 
of  the  correctness  of  Weissenfeld's  conclusions.  This 
property  is  so  variable  as  to  have  no  important  value. 
Colon  bacilli  freshly  isolated  from  the  intestine  are 
frequently  non- virulent,  and  Savage  (1903*)  and  others 
have  shown  that  there  is  in  general  no  correlation 
between  pathogenic  power  and  direct  or  indirect  intes- 
tinal origin.  On  the  other  hand  Weissenfeld's  work 
entirely  fails  to  show  that  the  colon  bacillus,  pathogenic 
or  non-pathogenic,  is  a  normal  inhabitant  of  unpolluted 
waters.  Even  his  own  results,  if  the  quantitative  rela- 


146       ELEMENTS  OF  WATER  BACTERIOLOGY 

tions  be  considered,  furnish  evidence  to  the  contrary. 
In  24  of  the  26  samples  from  bad  sources,  he  isolated 
his  imperfectly  defined  colon  bacilli  from  i  c.c.  of  the 
water,  while  in  only  8  of  the  30  samples  of  good  waters 
could  he  find  such  organisms  in  that  quantity. 

Colon  Bacilli  on  Plants  and  Plant  Products.  The 
work  of  certain  recent  observers  has  suggested  the 
possibility  that  the  colon  bacillus  may  live  in  a  semi- 
parasitic  fashion  on  plants  as  well  as  on  animals.  Of 
a  series  of  47  cultures  of  lactic-acid  bacteria,  recently 
examined  by  one  of  ourselves  (Prescott,  i902a;  Prescott, 
1903,  Prescott,  1906),  25  were  found  to  give  the  reactions 
of  B.  coli.  These  organisms  were  isolated  chiefly  from 
cereals  and  products  of  milling,  such  as  flour,  bran, 
cornmeal,  oats,  barley,  etc.,  while  others  were  in  technical 
use  for  producing  the  lactic  fermentation.  There  is 
no  evidence  that  any  of  these  organisms  were  of  intesti- 
nal origin,  and  yet  they  possess  all  the  characters  of  typi- 
cal colon  bacilli,  even  to  the  pathogenic  action  when 
inoculated  into  guinea  pigs.  In  Germany,  Papasotiriu 
(Papasotiriu,  1901)  was  meanwhile  carrying  on  almost 
exactly  similar  investigations  to  Prescott's,  with  identi- 
cal results. 

Other  testimony  is  somewhat  conflicting  with  regard 
to  the  occurrence  of  B.  coli  on  plants.  Klein  and 
Houston  (1900)  reported  the  finding  of  typical  colon 
bacilli  in  only  3  out  of  24  samples  of  wheat  and  oats 
obtained  from  a  wholesale  house;  rice,  flour,  and  oat- 
meal bought  at  two  different  retail  shops  gave  B.  coli 
in  all  three  cereals  in  one  case  and  on  none  in  the  other. 
Clark  and  Gage  (1903)  were  unable  to  isolate  B.  coli 


SIGNIFICANCE  OF  COLON  GEOUP  IN  WATER     147 

from  standing  grains.  Gordan  (1904)  could  not  find 
B.  coli  in  .1  and  .01  mg.  samples  of  clean  bran,  but 
isolated  it  easily  from  that  of  poor  quality.  Winslow 
and  Walker  (1907)  have  recently  reported  the  examina- 
tion of  178  samples  of  grain  and  40  samples  of  grasses 
for  B.  coli  without  success.  On  the  other  hand,  Diiggeli 
(1904)  found  B.  coli  among  the  bacteria  occurring  on 
the  leaves  of  growing  plants,  although  it  was  not  one 
of  the  most  abundant  species.  Barthel,  too  (Barthel, 
1906),  found  B.  coli  widely  distributed  on  plants  from 
both  cultivated  and  uncultivated  regions.  Bettencourt 
and  Borges  (1908^  examined  35  samples  of  vegetables 
and  cereals  purchased  in  open  market  and  found  12 
lactose-fermenting  forms,  of  which  only  6  proved  to  be 
B.  coli.  It  should  be  noted,  however,  that  the  method 
of  isolation  used  was  direct  plating  on  Endo-medium, 
which  is  of  course  less  sensitive  than  the  enrichment 
processes  used  by  other  workers. 

Neumann  (1910)  has  recently  studied  the  distribu- 
tion of  colon  bacilli  on  and  in  various  food  substances 
such  as  bread,  milk,  butter  and  fruit.  From  fresh 
fruits  immediately  after  picking  he  never  isolated 
them,  but  they  were  present  in  a  certain  proportion  of 
all  the  foods  which  had  been  exposed  to  human  con- 
tamination and  the  author  concludes  that  wherever 
human  hands  have  been,  there  will  B.  coli  be  found. 
Konrich  (1910)  in  a  similar  series  of  investigations 
obtained  positive  results  from  46  out  of  100  .1  to  .5  gm. 
samples  of  cultivated  plants  while  leaves  of  trees  and 
grasses  and  herbs  on  waste  places  gave  about  6  per 
cent  positive  results.  Hay  showed  colon  bacilli  in 


148       ELEMENTS  OF  WATER  BACTERIOLOGY 

91  per  cent  of  the  135  samples  examined  and  grains 
in  55  per  cent  of  300  samples. 

Colon  Bacilli  in  Dust  and  Soil.  Winslow  and  Kligler 
(1912)  have  shown  that  colon  bacilli  may  be  very 
abundant  in  the  dust  of  city  streets  and  houses,  as 
might  naturally  be  expected  from  the  fact  that  such 
dust  is  largely  made  up  of  horse  droppings.  They 
examined  24  samples  of  street  dust  and  72  samples  of 
house  dust  (all  in  New  York  City).  All  of  the  street 
dusts  and  63  of  the  72  house  dusts  contained  colon 
bacilli  in  at  least  one  of  three  duplicate  i<U  gram 
portions.  In  two  street  samples  the  numbers  rose  to 
330,000  and  660,000  per  gram  respectively,  while  the 
largest  indoor  result  was  60,000.  The  average  for  the 
indoor  dusts  was  between  1000  and  2000  per  gram  and 
for  the  street  dusts  over  50,000  per  gram.  This  dust 
was  dust  deposited  on  surfaces  and  would  only  be 
carried  up  into  the  air  by  currents  of  some  force.  It 
is  well  known  that  colon  bacilli  are,  as  a  matter  of  fact, 
rarely  present  in  street  or  house  air.  Konrich  (1910) 
exposed  open  Petri  dishes  of  dextrose  broth  to  the  air 
of  Jena  streets  for  24-hour  periods,  daily,  for  3  months 
and  found  colon  bacilli  only  n  times.  The  colon 
bacilli  in  street  dust  may,  however,  perhaps  account 
for  the  anomalous  positive  results  sometimes  obtained 
in  reservoirs  bordered  by  roadways. 

Konrich  (1910)  has  also  made  important  contribu- 
tions to  the  study  of  colon  bacilli  in  the  earth.  Out 
of  547  samples  of  soil,  65%  showed  B.  coli  in  por- 
tions of  between  .1  and  .5  gm.  The  farther  removed 
from  cultivation  a  sample  was,  the  less  were  the  chances 


SIGNIFICANCE  OF  COLON  GROUP  IN  WATER     149 

of  positive  results.  He  concludes  that  B.  coli  is  widely 
distributed  in  the  outer  world.  It  is  almost  always 
found  in  soil  from  cultivated  fields  or  from  traveled 
places.  The  farther  a  source  is  removed  from  travel 
and  from  cultivation  the  more  rarely  is  the  colon 
bacillus -found;  but  it  is  never  altogether  absent.  On 
plants  or  parts  of  plants  it  is  frequently  found  when  they 
come  from  cultivated  land;  on  plants  from  waste  places 
it  is  rarely  found.  It  seems  probable  that  colon  bacilli 
may  be  even  more  widely  distributed  in  a  thickly 
settled  and  intensively  cultivated  country  like  Ger- 
many than  in  the  United  States. 

The  Number  of  Colon  Bacilli,  not  Their  Mere  Pres- 
ence, as  an  Index  of  Water  Pollution.  The  more 
important  practical  conclusions  to  be  drawn  from  these 
various  investigations  seem  to  be  as  follows: 

1.  Bacteria  corresponding  in  every  way  to  B.  coli  are 
by  no   means   confined   to   animal   intestines,   but  are 
widely  distributed  elsewhere  in  nature. 

2.  The  finding  of  a  few  colon  bacilli  in  large  samples 
of  water,  or  its  occasional  discovery  in  small  samples, 
does  not  necessarily  have  any  special  significance. 

3.  The  detection  of  B.  coli  in  a  large  proportion  of 
small  samples  (i  c.c.  or  less)  examined  is  imperatively 
required   as  an   indication   of  recent   sewage   pollution. 

4.  The  number  of  colon  bacilli  in  water  rather  than 
their  presence  should  be  used  as  a  criterion  of  recent 
sewage  pollution. 

With  these  qualifications  the  value  of  the  colon  test 
was  never  more  firmly  established  than  it  is  to-day. 
Whether  or  not  originally  a  domesticated  form,  it  is 


150       ELEMENTS  OF  WATER  BACTERIOLOGY 

clear  that  the  colon  bacillus  finds  in  the  intestine  of  the 
higher  vertebrates  an  environment  better  suited  to  its 
growth  and  multiplication  than  any  other  which  occurs 
in  nature.  Houston  (igof)  records  the  number  of  B. 
coli  per  gram  of  normal  human  faeces  as  between 
100,000,000  and  1,000,000,000.  It  is  almost  certain 
that  the  only  way  in  which  large  numbers  of  these 
organisms  gain  access  to  natural  waters  is  by  pollution 
with  the  domestic,  industrial,  and  agricultural  wastes 
of  human  life.  If  pollution  has  been  recent,  colon 
bacilli  will  be  found  in  comparative  abundance.  If 
pollution  has  been  remote  the  number  of  colon  bacilli 
will  be  small,  since  there  is  good  evidence  that  the 
majority  of  intestinal  bacteria  die  out  in  water.  If 
derived  from  cereals  or  the  intestines  of  wild  animals,  the 
number  will  be  insignificant  except  perhaps  in  the  vicinity 
of  great  grain-fields  or  where  the  water  receives  refuse 
from  grist-mills,  tanneries,  dairies,  or  lactic-acid  factories. 
The  first  recognition  of  the  necessity  for  a  quantita- 
tive estimation  of  colon  bacilli  in  water  we  owe  to  Dr. 
Smith,  who  in  1892  (Smith,  1893^  outlined  a  plan  for  a 
study  to  be  made  by  the  New  York  Board  of  Health  on 
the  Mohawk  and  Hudson  Rivers.  Burri  (Burri,  1895) 
pointed  out  that  the  use  of  so  large  a  sample  as  a  liter 
for  examination  would  lead  to  the  condemnation  of 
many  good  waters.  Freudenreich  (Freudenreich,  1895) 
at  the  same  time  indicated  the  necessity  for  taking  into 
account  the  number  of  colon  bacilli  present.  He  recorded 
the  isolation  of  the  organisms  from  unpolluted  wells, 
when  as  large  a  quantity  of  water  as  100  c.c.  was  used, 
and  concluded  that  it  was  entirely  absent  only  from 


SIGNIFICANCE  OF  COLON  GROUP  IN  WATER     151 

waters  of  great  purity  and  present  in  large  numbers 
only  in  cases  of  high  pollution.  This  author  also 
quoted  Miquel  as  having  found  colon  bacilli  in  almost 
every  sample  of  drinking-water  if  only  a  sufficient  por- 
tion were  taken  for  analysis. 

The  practical  results  of  the  application  of  the  colon 
test  from  this  standpoint  have  proved  of  the  highest 
value.  As  originally  outlined  by  Dr.  Smith,  it  con- 
sisted in  the  inoculation  of  a  series  of  dextrose  tubes 
with  small  portions  of  water,  tenths  or  hundredths  of 
the  cubic  centimeter.  It  was  first  used  by  Brown 
(Brown,  1893)  in  1892  for  the  New  York  State  Board 
of  Health,  and  it  showed  from  22  to  92  faecal  bacteria 
per  c.c.  in  the  water  of  the  Hudson  River  at  the  Albany 
intake,  and  from  3  to  49  at  various  points  in  the  Mohawk 
River  between  Amsterdam  and  Schenectady.  In  some 
previous  work  at  St.  Louis,  the  colon  bacilli  in  the 
Mississippi  River  were  found  to  vary  from  3  to  7  per  c.c. 

Hammerl  (Hammerl,  1897)  used  the  presence  of 
Bacillus  coli  as  a  criterion  of  self-purification  in  the 
river  Mur.  He  considered,  in  spite  of  the  position  taken 
by  Kruse,  that  when  a  water  contained  large  numbers 
of  colon  bacilli,  as  well  as  an  excess  of  bacteria  in 
general,  it  might  be  considered  to  be  contaminated  by 
human  or  animal  excrement.  As,  however,  the  organism 
would  naturally  be  present  in  large  quantities  of  such 
a  water  as  that  of  the  Mur,  he  used  no  enrichment 
process,  but  made  plate  cultures  direct;  he  defined  the 
B.  coli  as  a  small  bacillus,  non-motile  or  but  feebly 
motile,  growing  rapidly  at  37°  C.,  coagulating  milk 
and  forming  gas  in  sugar  media.  In  general,  Hammerl 


152       ELEMENTS  OF  WATEE  BACTEEIOLOGY 

failed  to  find  colon  bacilli  in  the  river  by  this  method, 
except  immediately  below  the  various  towns  situated 
upon  it;  at  these  points  of  pollution  he  discovered  a 
few  colon  colonies  upon  his  plates,  not  more  than  4  to 
6  per  c.c.  of  the  water.  He  concluded  that  "  the 
Bacterium  coli,  even  when  it  is  added  to  a  stream  in 
great  numbers,  under  certain  circumstances  disappears 
very  rapidly,  so  that  it  can  no  longer  be  detected  in 
the  examination  of  small  portions  of  the  water." 

The  most  important  work  upon  the  distribution  of  B. 
coli  has  been  that  carried  out  in  England  by  the  bacteri- 
ologists of  the  local  government  board,  by  Dr.  Houston 
in  particular.  This  investigator  (Houston,  1898;  Hous- 
ton, 1899*;  Houston,  igooa)  made  an  elaborate  series 
of  examinations  of  soils  from  various  sources  to  see 
whether  the  microbes  considered  to  be  characteristic 
of  sewage  could  gain  access  to  water  from  surface  wash- 
ings free  from  human  contamination.  In  the  three 
papers  published  on  this  subject  the  examination  of  46 
soils  was  recorded.  In  only  10  of  the  samples  was  B.  coli 
found,  and  of  these  10,  9  were  obviously  polluted,  being 
derived  from  sewage  fields,  freshly  manured  land,  or 
the  mud-banks  of  sewage-polluted  rivers.  The  author 
finally  concluded  that  "  as  a  matter  of  actual  observa- 
tion the  relative  abundance  of  B.  coli  in  pure  and  impure 
substances  is  so  amazingly  different  as  to  lead  us  to 
suspect  that  not  only  does  B.  coli  not  flourish  in  nature 
under  ordinary  conditions,  but  that  it  tends  to  even 
lose  its  vitality  and  die."  "  In  brief,  I  am  strongly 
of  opinion  that  the  presence  of  B.  coli  in  any  number, 
whether  in  soil  or  in  water,  implies  recent  pollution  of 


SIGNIFICANCE  OF  COLON  GROUP  IN  WATER     153 

animal  sort."  Pakes  (Pakes,  1900)  stated  on  the 
strength  of  an  examination  of  "  about  300  different 
samples  of  water,"  no  particulars  being  published,  that 
water  from  a  deep  well  should  not  contain  B.  coli  at 
all,  but  that  water  from  other  sources  need  not  be  con- 
demned unless  the  organism  was  found  in  20  c.c.  or  less. 
When  colon  bacilli  were  found  only  in  greater  quantities 
than  100  c.c.  the  water  might  be  considered  as  probably 
safe.  Horrocks  (Horrocks,  1910),  after  a  general 
review  of  English  practice,  concluded  that  "  when  a 
water-supply  has  been  recently  polluted  with  sewage, 
even  in  a  dilution  of  one  in  one  hundred  thousand,  it 
is  quite  easy  to  isolate  the  B.  coli  from  i  c.c.  of  the 
water."  "  I  would  say  that  a  water  which  contained 
B.  coli  so  sparingly  that  200  c.c.  required  to  be  tested 
in  order  to  find  it  had  probably  been  polluted  with 
sewage,  but  the  contamination  was  not  of  recent  date." 
Chick  (Chick,  1900)  found  6100  colon  bacilli  per  c.c. 
in  the  Manchester  ship  canal,  55-190  in  the  polluted 
River  Severn,  and  numbers  up  to  65,000  per  gram 
in  roadside  mud.  On  the  other  hand,  of  38  unpolluted 
streams  and  rivulets,  31  gave  no  Bacillus  coli  and  the 
other  7  gave  i  per  c.c.  or  less.  The  Liverpool  tap  water, 
snow,  rain,  and  hail  showed  no  colon  bacilli. 

One  of  the  first  elaborate  applications  of  the  colon 
test  was  made  by  Jordan  in  the  examination  of  the  fate 
of  the  Chicago  sewage  in  the  Desplaines  and  Illinois 
Rivers.  In  these  studies  of  self-purification  (Jordan, 
1901)  the  analyses  were  made  quantitative  by  the 
examination  of  numerous  measured  samples,  fractions 
of  the  cubic  centimeter;  and  the  method  employed 


154       ELEMENTS  OF  WATER  BACTERIOLOGY 

was  enrichment,  either  in  dextrose-broth  fermentation 
tubes  or  in  phenol  broth,  with  subsequent  plating 
on  litmus  lactose  agar.  The  cultures  isolated  were 
tested  as  to  their  behavior  in  dextrose  broth,  pep- 
tone solution,  milk,  and  gelatin;  of  the  dextrose 
tubes  made  directly  from  the  water  all  were  con- 
sidered positive  which  gave  more  than  20  per  cent 
gas  in  the  closed  arm,  with  an  appreciable  excess  of 
hydrogen.  The  results  were  very  significant.  In  fresh 
sewage  a  positive  result  was  obtained  about  one-third  of 
the  time  in  one  one-hundred-thousandth  of  a  cubic  cen- 
timeter and  almost  constantly  in  one-ten-thousandth 
of  a  cubic  centimeter.  The  Illinois  and  Michigan 
canal  proved  almost  as  bad,  giving  positive  results  on  7 
days  out  of  28  in  dilutions  of  one  in  a  hundred  thousand 
and  on  28  days  out  of  32  in  a  dilution  of  one  in  ten 
thousand.  At  Morris,  27  miles  below  Lockport,  where 
the  canal  enters  the  bed  of  the  Desplaines  River,  and 
9  miles  below  the  entrance  of  the  Kankakee,  the 
principal  diluting  factor,  the  numbers  were  so  reduced 
that  positive  results  were  obtained  only  on  n  days  out 
of  twenty  in  one-thousandth  of  a  cubic  centimeter,  on 
20  days  out  of  thirty  in  one-hundredth  of  a  cubic  centi- 
meter, and  on  20  days  out  of  23  in  one-tenth  of  a  cubic 
centimeter.  At  Averyville,  159  miles  below  Chicago, 
colon  bacilli  were  isolated  on  only  4  days  out  of  27  in 
one- tenth  of  a  cubic  centimeter,  and  on  13  days  out  of 
31  in  one  cubic  centimeter.  A  comparison  with  certain 
neighboring  rivers  showed  this  to  be  about  the  normal 
value  for  waters  of  similar  character,  as  the  following 
table  extracted  from  Professor  Jordan's  paper  will  show : 


SIGNIFICANCE  OF  COLON  GEOUP  IN  WATER     155 


NUMBER  OF  B.  COLI  PRESENT  IN  CERTAIN  RIVER 
WATERS 

(JORDAN,  1901) 


O.I 

C.C. 

I   C 

.c. 

Source  of  Sample. 

No.  Days 
Water 
Examined. 

No.  Days 
B.  Coli 
Found. 

No.  Days 
Water 
Examined. 

No.  Days 
B.  Coli 
Found. 

Illinois  River,  Averyville  .  .  . 
Mississippi  River,  Grafton  .  . 
Fox  River 

27 
34 

22 

4 
10 
2 

31 
35 

23 

13 
23 
6 

Sangamon  River  

2< 

14 

27 

21 

Missouri  River 

12 

17 

31 

21 

These  results  harmonize  rather  closely  with  those 
previously  recorded  by  Brown  and  Fuller  and  indicate 
that  in  the  larger  rivers  where  the  proportionate  pollu- 
tion is  not  extreme,  colon  bacilli  may  be  isolated  in  about 
half  the  i-c.c.  samples  examined.  Such  rivers  are  of 
course  inadmissible  as  sources  of  water-supply,  accord- 
ing to  modern  sanitary  standards,  unless  subjected  to 
purification  of  some  sort. 

Hunnewell  and  one  of  us  (Winslow  and  Hunnewell, 
igo2b)  examined  a  considerable  series  of  normal  waters 
for  B.  coli,  testing  i  c.c.  from  each  by  the  dextrose- 
broth  method  and  a  larger  portion  of  100  c.c.  by  incuba- 
tion with  phenol  broth  as  described  in  Chapter  VI. 
The  samples  were  obtained  from  the  public  supplies  of 
Taunton,  Boston,  Cambridge,  Braintree,  Brookline, 
Needham,  and  Lynn  in  Massachusetts,  and  Newport, 
R.  I.,  from  the  Sudbury  River,  from  the  ocean,  from 
the  waters  of  springs  bottled  for  the  market,  from 
ponds,  pools  of  rain  and  melted  snow,  springs,  brooks, 
shallow  wells,  and  driven  wells  in  various  towns  near 


156       ELEMENTS  OF  WATEE  BACTERIOLOGY 


the  city  of  Boston.  For  comparison  50  samples  of 
polluted  waters  from  the  Charles,  Mystic,  Neponset, 
and  North  Rivers  were  examined.  The  colon  bacillus 
was  defined  by  gas  production  in  dextrose  broth, 
coagulation  of  milk,  reduction  of  nitrates,  formation 
of  indol  and  failure  to  liquefy  gelatin;  and  organisms 
which  lacked  the  power  to  reduce  nitrates  or  to  form 
indol  were  classed  in  the  "  Paracolon  group."  The 
results  are  summarized  in  the  following  table: 

PRESENCE    OF  B.  COLI  IN  POLLUTED  AND  UNPOLLUTED 
WATERS 

(WlNSLOW  AND  HUNNEWELL,  IQO2b) 

Unpolluted  Waters 


Samples  examined 157 

Dextrose  broth  positive 40 

Lactose  plate  positive 13 

Colon  group 5 

Paracolon  group 5 

B.  cloacae  group 

Streptococcus  group 3 

Polluted  Waters 

I    C.C. 

Samples  examined 50 

Dextrose  broth  positive 50 

Lactose  plate  positive 50 

Colon  group 18 

Paracolon  group 6 

B.  cloacae i 

Streptococcus  group 25 


76 

3i 
ii 

5 

5 

10 


48 

37 
26 


As  the  authors  pointed  out,  these  tables  indicate  that 
bacteria  capable  of  growth  at  the  body  temperature  and 
fermenting  dextrose  and  lactose  are  infrequently  found 


SIGNIFICANCE  OF  COLON  GROUP  IN  WATER     157 


in  unpolluted  waters,  and  colon  bacilli  are  very  rarely 
present.  In  157  samples,  typical  colon  bacilli  were 
found  only  5  times  out  of  157,  in  i  c.c.  Lactose  ferment- 
ing organisms  appeared  in  only  8  per  cent  of  the  nor- 
mal samples  and  in  100  per  cent  of  the  polluted  ones, 
in  i  c.c.  Incidentally  it  may  be  pointed  out  that  these 
tables  well  illustrated  the  dangers  of  overgrowths, 
particularly  in  large  samples.  It  is  clear  that  the  strep- 
tococci had  killed  out  colon  bacilli,  originally  present, 
in  a  large  proportion  of  the  loo-c.c.  samples  of  polluted 
waters  and  in  some  of  the  i-c.c.  samples,  since,  in  so 
many  cases,  gas  formation  was  followed  by  the  isola- 
tion of  the  streptococcus  alone. 

Colon  Bacilli  in   Surface   Waters.     Clark   and  Gage 
(1903)  have  published  the  results  of  certain  studies  of 


DISTRIBUTION  OF    TOTAL    BACTERIA 
SURFACE-WATERS 

(CLARK  AND  GAGE.  1903) 


AND    B.    COLI     IN 


Lake. 

Population  of 
Watershed  per 
Square  Mile. 

Bacteria 
per  c.c. 

B.  Coli 
Per  Cent  Positive  Tests. 

I    C.C. 

100  C.C. 

I* 

1400 

612 

13-3 

33-o 

2 

356 

319 

3-5 

17.2 

3 

116 

103 

0.0 

o.o 

4 

9° 

170 

o.o 

14.0 

5 

62 

87 

o.o 

9.0 

6* 

60 

48 

2-3 

4-5 

7* 

50 

66 

4-6 

21  .0 

8 

47 

133 

0.0 

9.0 

9 

42 

J3i 

0.0 

6.7 

10* 

40 

31 

0.0 

6.2 

ii 

8 

28 

0.0 

7-7 

12 

42 

107 

0.0 

9-3 

*  Shores  used  for  pleasure  resorts. 


158       ELEMENTS  OF  WATER  BACTERIOLOGY 


Massachusetts  ponds  which  indicate  clearly  the  coin- 
cidence of  the  distribution  of  B.  coli  in  single  centimeters 
of  surface  waters,  with  actual  sanitary  conditions. 
They  show  also  the  slight  significance  of  the  test  for 
this  organism  in  larger  volumes  of  water.  Almost 
every  source  gave  positive  tests  in  100  c.c.,  while  with 
i-c.c.  samples  only  those  lakes  appear  suspicious  which 
are,  in  fact,  exposed  to  dangerous  pollution. 

Houston  (1905)  gives  the  following  table,  which  may 
be  taken  as  another  fair  example  of  the  distribution  of  B. 
coli  in  small  streams  and  lakes.  Of  the  two  lakes 
studied,  Loch  Ericht  is  free  from  the  pollution  of  human 
or  domesticated  animals,  while  Loch  Laggan  receives 
some  drainage  from  farm  lands;  both  are  of  large 
size.  The  brook  and  river  samples  were  collected  from 
adjacent  streams. 

DISTRIBUTION  OF  B.  COLI  IN  SURFACE-WATERS 

(HOUSTON,  1905) 
Percentage  of  Samples  showing  B.  Coli  in  each  Dilution. 


Dilution. 

+  O.I   C.C. 

+  1.0  C.C. 
—  O.I   C.C. 

+  10  C.C. 
—  I   C.C. 

+  IOO  C.C. 
—  IO  C.C. 

Not  in 

IOO  C.C. 

Brooks  and  river. 
Loch  Laggan. 

7-7 

53.8 

I  .  2 

34-6 

33  -O 

3.8  • 

49  -4 

16  .4 

Loch  Ericht  

I  .0 

19  .O 

80.0 

As  an  example  of  a  heavily  polluted  stream,  011  the 
other  hand,  the  table  on  page  159  may  be  cited.  It  shows 
in  a  striking  way  the  increase  of  B.  coli  in  the  Thames 
on  its  passage  through  London  and  its  progressive 
purification  below. 


SIGNIFICANCE  OF  COLON  GROUP  IN  WATER    159 


The  river  at  the  lower  stations  in  this  table  was  con- 
siderably diluted  with  sea-water,  yet  it  showed  clearly 
its  large  proportion  of  sewage.  Normal  sea- water, 
even  in  the  neighborhood  of  the  shore,  shows,  B.  coli 
only  in  large  samples.  Houston  (1904),  in  another 
communication,  reports  the  examination  of  168  samples 
of  sea- water  near  the  English  coast.  None  of  the  samples 
showed  B.  coli  in  i  c.c.;  97  samples  gave  negative 
results  in  10  c.c.;  45  in  100  c.c.,  and  4  had  no  B.  coli 
even  in  1000  c.c. 

B.    COLI    IN   THE    RIVER   THAMES    AT   VARIOUS    POINTS 

(HOUSTON,  i904a) 
Percentage  of  Positive  Results 


Place. 

—  10 

c.c. 

+  10 
—  I   C.C. 

+  i 

—  O.I 

c.c. 

+  .1 

—  O.OI 

c.c. 

+    .01 
—  0.001 

c.c. 

+  0.001 
—  0.0001 

c.c. 

+0.0001 
—  O.OOOOI 

c.c. 

Sunbury 

70  6 

23     < 

t;  Q 

Hampton. 

ii.  8 

64  7 

177 

c  0 

Barking  

4.2 

45-8 

45-8 

4.2 

Crossness 

ii   i 

27    7 

CQ   O 

II    I 

Purfleet  

3.0 

9.1 

33-3 

39-i 

15-1 

Grays    

2.8 

22  .  2 

41  .  7 

77  .  7 

Mucking 

30  8 

C7   7 

ii   S 

Chapman  

C    O 

4<  o 

^O.O 

Barrow  Deep  .  . 

12.  O 

36.0 

40.0 

12.0 



Gartner  (1910)  has  collected  some  interesting  data 
in  regard  to  the  ratio  between  the  number  of  colon 
bacilli  and  of  total  bacteria  in  waters  of  different 
quality.  The  results  from  four  different  sets  of  experi- 
ments by  Konrich  at  Jena,  Houston  at  London,  Noble 
in  New  York,  and  Hill  at  Giessen,  may  be  combined 
as  on  the  opposite  page: 


160       ELEMENTS  OF  WATER  BACTERIOLOGY 


RATIO    OF    TOTAL    BACTERIA    TO    COLON    BACILLI    IN 
WATERS  OF  DIFFERENT   CLASSES 


Coli  titer— 
Smallest  Por- 
tion of  Water 
Showing 
B.  Coli. 

Ratio  of  Plate  Count  to  B.  Coli. 

Jena. 

20° 

London. 

New  York. 

Giessen. 

20°. 

37°. 

24°. 

37°. 

IOO  C  C 

60,500 
15,100 
1,288 

352 
46 

1  1,  800 

1,810 
255 
34 
2.4 

1950 
213 
2O 

280 
47 
7 

87 

20 

4-9 
0.4 

10  C.C  
I  C.C  
O.I  C.C  
O.OI  C.C  
O  OOI  C  C 

695 
183 
29.7 
4.1 

O.OOOI  C.C. 

O.OOOOI  C.C..  . 



The  rather  regular  decrease  in  the  ratio  of  the  total 
count  to  the  B.  coli  count  with  an  increase  in  the  actual 
number  of  colon  bacilli  is  very  interesting. 

Prof.  Gartner  apparently  holds  that  this  fall  in  the 
ratio  of  the  plate  count  to  the  u  coli  titer  "  indicates  a 
fallacy  in  the  method  of  the  latter  and  in  particular  he 
emphasizes  the  absurdity  of  the  lowest  figures  in  the  table 
which  indicates  that  there  were  twice  as  many  colon 
bacilli  as  bacteria  of  all  sorts.  It  seems  to  us  that  the 
last  phenomenon  is  quite  as  likely  to  be  due  to  an  error  in 
the  plate  count  as  to  a  failure  in  the  enrichment  pro- 
cedure. Unless  dilutions  are  very  carefully  made 
plates  inoculated  with  waters  containing  tens  and 
hundreds  of  thousands  of  bacteria  per  c.c.  are  pretty 
likely  to  be  so  crowded  that  only  a  portion  of  the  bac- 
teria with  which  they  are  sown  are  able  to  develop. 
As  to  the  diminishing  ratio  with  increasing  coli-content, 
it  is  exactly  what  might  reasonably  be  expected.  One- 
tenth  to  one-quarter  of  the  bacteria  in  sewage  may  be 


SIGNIFICANCE  OF  COLON  GROUP  IN  WATER     161 

colon  bacilli,  and  the  greater  the  amount  of  sewage 
in  water,  the  nearer  this  ratio  will  be  approached. 

Colon  Bacilli  in  Ground- waters.  With  ground-waters 
the  story  is  the  same.  Even  in  sources  of  excellent 
quality  we  should  expect  to  find,  and  we  do  sometimes 
find,  colon  bacilli  in  large  volumes  of  water.  Abba, 
Orlandi,  and  Rondelli  (1899)  showed  by  experiments 
with  B.  prodigiosus  at  Turin  that  when  bacteria  are 
present  in  great  numbers  on  the  surface  of  the  ground, 
a  few  may  penetrate  for  a  considerable  distance  and 
ultimately  reach  the  sources  of  ground-waters.  The 
chance  that  disease  germs  could  survive  this  process  in  a 
soil  so  impervious  as  to  allow  colon  bacilli  to  appear 
only  in  large  samples  of  water,  is  infinitesimal. 

An  interesting  contribution  to  the  bacteriology  of 
ground-waters  was  made  by  the  Massachusetts  State 
Board  of  Health  (Massachusetts  State  Board  of  Health, 
1901)  in  connection  with  the  examination  of  the  spring- 
waters  bottled  for  the  sale  in  the  State.  Ninety-nine 
springs  were  included  in  this  study,  and  in  almost  every 
instance  4  samples  were  examined,  2  taken  directly 
from  the  spring  by  the  engineers  of  the  board  and  2 
from  the  bottles  as  delivered  for  sale  to  the  public.  In 
the  water  of  one  spring  B.  coli  was  found  twice,  once 
in  a  sample  from  the  spring  and  once  in  the  bottled 
sample.  This  spring  was  situated  in  woodland,  but 
was  unprotected  from  surface  drainage,  and  the  method 
of  filling  bottles  subjected  it  to  possible  contamination. 
In  5  other  cases  B.  coli  was  found  once  in  the  sample 
from  the  spring;  all  were  subject  to  pollution  from 
dwellings  or  cultivated  fields,  and  4  of  the  5  were  shown 


162       ELEMENTS  OF  WATER  BACTERIOLOGY 


to  be  highly  contaminated,  chemically.  In  7  other 
cases  B.  coli  was  found  in  the  bottled  samples  alone; 
3  of  these  sources  were  of  high  purity,  but  the  bottling 
process  furnished  opportunity  for  contamination. 

Clark  and  Gage  (1903),  in  the  examination  of  170 
samples  of  water  from  tubular  and  curb  wells  of  good 
quality  used  as  sources  of  water-supply,  found  B.  coli 
only  5  times,  once  in  i  c.c.  and  4  times  in  100  c.c.  Horton 
(1903),  from  a  study  of  ground-waters  in  Ohio,  concluded 
that  the  presence  of  B.  coli  in  wells  and  springs  was 
indicative  of  serious  pollution. 

Houston  (i903b)  makes  an  instructive  comparison  of 
some  more  or  less  polluted  shallow  wells  at  Chichester 
with  deep  ground-waters  of  high  quality  at  Tunbridge 
Wells.  The  following  table  shows  the  value  of  the 
i  cubic-centimeter  sample  in  discriminating  between 
good  and  bad  waters. 

DISTRIBUTION   OF   B.    COLI   IN   GOOD   AND    BAD   WELL 
WATERS 

(HOUSTON,  1903^ 
Percentage  of  Positive  Tests 


Quantity  of  Water. 

Chichester  Shallow 
Wells. 

Tunbridge  Wells, 
Deep  Wells. 

IOO  C.C. 
IO  C.C. 

90 
80 

25 
6 

I  C.C. 

45 

O 

O.  I   C.C. 

20 

0 

In  a  subsequent  investigation,  Houston  (1905)  exam- 
ined still  larger  samples  of  water  from  the  Tunbridge 
Wells  for  B.  coli:  49  samples  of  100  c.c.  each  showed  no 
B.  coli,  and  27  liter  samples  showed  B.  coli  only  once. 


SIGNIFICANCE  OF  COLON  GROUP  IN  WATER     163 

Kaiser  (1905)  reports  an  interesting  correlation  between 
total  numbers  and  B.  coli  in  a  series  of  38  well  waters. 
Of  1 1  wells  containing  over  200  bacteria  per  c.c.  90  per  cent 
showed  colon-like  organisms  in  liter  samples.  Of  1 2  wells 
containing  from  50  to  200  bacteria  per  c.c.  67  per  cent 
gave  colon-like  organisms;  of  26  wells  with  le  s  than  50 
bacteria  per  c.c.,  only  27  per  cent  showed  positive  results. 
Fromme  (1910)  brings  out  the  relation  between  B. 
coli  and  total  numbers  in  120  samples  of  well  waters 
near  Hamburg  in  the  table  below. 

RELATION  BETWEEN  TOTAL  NUMBERS  OF  BACTERIA 
AND    B.    COLI 

(FROMME,  1910) 


Colony  Count. 

Number  of  Samples. 

Per  cent  Positive 
B.  coli  Tests  in  10  c.c. 

Over  200 
50-200 
Under  50 

35 
19 
66 

40.0 
15-8 

Similar  data  obtained  by  one  of  us  for  some  American 
sources  have  been  cited  in  Chapter  I.  Even  Konrich 
(1910),  who  is  exceedingly  rceptical  as  to  the  value  of 
the  colon  test,  has  shown  that  an  increase  in  the  colon 
content  of  the  Jena  water  supply  (a  ground-water) 
always  followed  a  heavy  rain  which  washed  through 
some  of  the  colon  bacilli  in  the  soil. 

Colon  Bacilli  in  Filtered  Waters.  One  of  the  most 
important  applications  of  the  colon  test  is  in  the  control 
of  the  operation  of  municipal  water  niters.  It  has 
been  used  for  this  purpose  for  10  years  or  more  at 
Lawrence,  and  Fuller  laid  stress  upon  its  results  in  his 
classic  experiments  on  water  purification  in  the  Ohio 
valley.  At  Cincinnati  he  records  the  presence  of 


164       ELEMENTS  OF  WATER  BACTERIOLOGY 


colon  bacilli  in  60  per  cent  of  the  i-c.c.  samples  from  the 
Ohio  River,  while  the  effluent  from  either  slow  sand  or 
mechanical  niters  gave  positive  results  only  half  the 
time  in  samples  of  50  c.c.  The  results  of  the  examina- 
tions carried  out  at  Lawrence  for  6  years  are  brought 
together  in  the  table  below  from  the  Annual  Reports 
of  the  Massachusetts  State  Board  of  Health. 

B.   COLI   IN   MERRIMAC   RIVER  AND   LAWRENCE  FILTER 
EFFLUENT 


Merrimac  River,  Per 
cent  of  i  c.c., 
Samples  containing 
B.  coli. 

Merrimac  River, 
Number  B.  coli  per 
c.c. 

Filtered  Water,  Per 
cent  of  i  c.c., 
Sample  containing 
B.  coli. 

1900 

99-7 

8? 

iS.I 

1901 

* 

# 

* 

1902 

99.0 

73 

4.0 

1903 

99-0 

78 

4.2 

1904 

100.  0 

73 

8.0 

1905 

IOO.O 

118 

4-7 

*  Not  given. 

At  Harrisburg,  Pa.,  mechanical  nitration  combined 
with  chlorin  disinfection  has  yielded  the  results  tabulated 
below : 

B.  COLI  IN  RAW  AND  TREATED  WATER  AT 
HARRISBURG,   PA. 

(HARRISBURG,  1913) 


Per  Cent  Positive  Tests  in  i  c.c. 


Raw  Water. 

Treated  Water. 

1906 

71.9 

2.7 

1907 

64.0 

I  .O 

1908 

65-7 

I  .  I 

1909 

63-1 

1.0 

1910 

55-4 

O.  2 

1911 

77-3 

0.6 

1912 

46.9 

0.8 

SIGNIFICANCE  OF  COLON  GROUP  IN  WATER     165 


At  Washington  the  most  complete  slow  sand- 
filtration  plant  yet  constructed  has  yielded  the  results 
tabulated  below,  for  which  we  are  indebted  to  the 
courtesy  of  Mr.  F.  F.  Longley: 

B.   COLI  IN  POTOMAC  RIVER  AND  WASHINGTON  FILTER 
EFFLUENT 


Dalecarlia  Reservoir  Inlet. 

Filtered-  Water  Reservoir 
Outlet. 

1906. 

Samples. 

Samples. 

Number 

Number 

Tested.             _^ 

, 

Tested. 

, 

, 

IO   C.C. 

I   C.C. 

10  C.C. 

I.   C.C. 

February.  .  .  . 

iS                5 

3 

24 

0 

0 

March  

24 

12 

3 

27 

0 

o 

April  

18 

9 

6 

25 

I 

o 

May  

25                3 

i 

27 

o 

o 

June 

26                    o 

8 

26 

o 

o 

July  

20 

•y 

8 

9 

21 

I 

o 

August  

26 

21 

14 

27 

I 

I 

September.  .  . 

10 

4 

i 

25 

2 

o 

October  

10 

3 

2 

27 

I 

o 

November.  .  . 

8 

3 

0 

25 

2 

0 

December.  .  . 

9 

4 

4 

24 

2 

2 

1907 

January  

9 

5 

3 

26 

3 

3 

February.  .  .  . 

8 

2 

2 

23 

0 

o 

March  

8 

7 

4 

26 

0 

0 

April  

Q 

4 

i 

26 

I 

0 

May  

23 

21 

15 

26 

0 

0 

June  

2e 

20 

17 

2j- 

o 

o 

July  

26 

II  - 

8 

26 

0 

0 

August  

27 

13 

8 

27 

0 

0 

September.  .  . 

24 

15 

13 

25 

I 

0 

It  must  be  remembered  that  in  the  Washington  plant 
filtration  is  supplemented  by  thorough  sedimentation, 
preliminary  and  subsequent.  The  entire  credit  for  the 
good  effluent  obtained  is  not  therefore  due  to  the  filters. 
At  Lawrence  it  has  been  shown  that  removal  of  colon 


166       ELEMENTS  OF  WATER  BACTERIOLOGY 


bacilli  in  storage  reservoirs  and  pipe  systems  may  be 
considerable.  The  figures  obtained  in  1900  at  various 
points  in  the  distribution  system  may  be  cited  as  an 
example. 

PERCENTAGE     OF     SAMPLES     OF     WATER     CONTAINING 
B.   COLL     LAWRENCE,   MASS. 

(MASSACHUSETTS  STATE  BOARD  OF  HEALTH,  1901) 


Effluent  of 
Filter. 

Outlet  of 
Reservoir. 

Tap 
City  Hall. 

Tap  Experi- 
ment Station. 

In  i  c.c  

18 

9 

4 

2 

In  100  c.c.  .  .  . 

38 

23 

16 

16 

Fromme  (1910)  in  the  examination  of  a  filtered 
water  averaging  35  bacteria  per  c.c.  obtained  the 
following  results  in  various  quantities  of  water. 

PERCENTAGE   OF    SAMPLES   OF  WATER  CONTAINING 
B.  COLL 

(FROMME,  1910) 


Number  of  Samples. 

Volume  Examined. 

Per  Cent  Positive. 

101 

2OO  C.C. 

30-9 

412 
800 

10  C.C. 
I   C.C. 

2.9 
0.25 

In  regard  to  the  proportion  of  positive  colon  tests 
permissible  in  a  filter  effluent,  Clark  and  Gage  (Clark 
and  Gage,  1900)  reported  some  specially  instructive 
observations  made  when  certain  of  the  underdrains  of 
the  Lawrence  filter  were  relaid  in  the  autumn  of  1898. 
In  doing  this  work  the  sand  on  some  of  the  beds  was 
seriously  disturbed;  and  in  December,  after  the  work  was 
completed,  B.  coli  was  found  in  i  c.c.  of  the  filtered 


SIGNIFICANCE  OF  COLON  GROUP  IN  WATER     167 

effluent  in  72  per  cent  of  the  samples  examined.  In 
January  and  February  the  organisms  were  found  in 
54  per  cent  and  62  per  cent  of  the  samples,  respectively, 
while  in  March  the  number  fell  to  a  normal  value  of 
8  per  cent.  Corresponding  to  this  excess  of  B.  coli 
in  the  city  water,  there  were  12  cases  of  typhoid  fever 
in  December,  59  cases  in  January,  12  in  February, 
and  9  in  March,  all  during  the  early  part  of  the  month. 
The  authors  conclude  that  "  when  filtering  a  river- 
water  as  polluted  as  that  of  the  Merrimac,  it  is  safe 
to  assume  that  when  B.  coli  is  found  only  infrequently 
in  i  c.c.  of  the  effluent,  the  typhoid  germs,  necessarily 
fewer  in  number  and  more  easily  removed  by  the 
filter,  have  been  eliminated  from  the  water." 

The  results  of  the  daily  tests  carried  out  at  municipal 
filter  plants  are  frequently  expressed  in  monthly  or 
yearly  averages,  as  in  some  of  the  cases  quoted  above. 
It  must  be  remembered,  however,  that  averages  of 
this  sort  are  accepted  only  by  courtesy  and  with  the 
implied  assumption  that  conditions  are  approximately 
constant  during  the  period  averaged.  When  it  is 
said  that  an  acceptable  effluent  may  show  B.  coli  in 
3  or  4  per  cent  of  the  samples  tested  the  statement  is 
true  only  for  a  series  of  samples  collected  and  examined 
at  the  same  time.  If  in  a  given  month  3  per  cent  of 
the  i  c.c.  samples  tested  show  B.  coli,  the  effluent  may 
or  may  not  be  safe.  If  on  each  of  20  days  3  B.  coli 
or  thereabouts  were  present  in  100  c.c.  of  the  water 
it  is  probably  a  safe  one.  If  on  19  days  no  B.  coli 
were  present,  and  on  the  twentieth  day  100  c.c.  showed 
60  B.  coli,  the  average  result  would  be  the  same,  but 


168       ELEMENTS  OF  WATER  BACTERIOLOGY 

the  water  on  one  day  was  of  a  dangerous  character. 
With  properly  managed  filter  plants  marked  varia- 
tions do  not  occur  from  day  to  day  and  average  results 
are  generally  reliable.  It  is  wholly  misleading,  how- 
ever, to  compare  such  results  with  the  average  exami- 
nations of  an  unfiltered  surface  water.  With  surface 
waters  daily  variations  are  the  rule  and  a  low  monthly 
average  of  colon  tests  may  include  and  cover  up 
dangerous  and  significant  high  numbers  at  particular 
periods. 

Summary  of  American  and  Foreign  Opinion  as  to 
the  Value  of  the  Colon  Test.  The  general  results  of  the 
studies  of  the  colon  tests  which  have  now  been  carried 
out  in  great  numbers  all  over  the  world  may  be  sum- 
marized by  a  few  further  citations. 

In  America  the  fact  that  the  number  of  colon  bacilli 
in  a  water  measures  the  degree  of  its  pollution  is  now 
universally  accepted.  The  same  conclusion  has  been 
established  in  England  by  the  elaborate  investigations 
of  Houston  and  his  pupils.  Savage,  for  example, 
concluded  (Savage,  1902)  from  a  study  of  a  large  num- 
ber of  water  supplies  in  Wales  that  even  in  surface 
waters,  exposed  to  animal  contamination  from  adjacent 
grazing  grounds,  B.  coli  is  not  present  in  2  c.c.  unless 
other  pollution  is  present.  In  a  more  recent  review 
of  the  whole  subject,  the  same  author  (Savage,  1906) 
concludes  that  "  there  is  no  evidence  or  observations 
which  have  ever  shown  that  B.  coli,  reasonably  defined, 
is  present  in  any  numbers  in  sources  which  have  not 
been  exposed  to  some  form  of  faecal  contamination." 

In    Germany,    Petruschky    and    Pusch    (Petruschky 


SIGNIFICANCE  OF  COLON  GROUP  IN  WATER     169 

and  Pusch,  1903)  examined  a  considerable  series  of 
waters  from  different  sources  by  incubating  measured 
samples  with  equal  amounts  of  nutrient  broth  and  iso- 
lating upon  agar.  In  45  samples  of  well-waters  they 
found  B.  coli  7  times  in  .01  c.c.,  9  times  in  .1  c.c.,  and 
7  times  in  i  c.c.  In  the  other  22  cases  it  could  not  be 
found  in  i  c.c.  and  in  4  cases  not  in  100  c.c.  One  sample 
showed  it  only  in  600  c.c.  and  i  not  in  750  c.c.  Of 
29  river-waters,  only  2  failed  to  give  positive  results 
in  .1  c.c.  and  14  showed  B.  coli  in  .001  of  a  c.c.  or  less. 
In  sewage  the  number  varied  from  i  to  1,000,000  per 
c.c.  The  authors  conclude  that  a  quantitative  estima- 
tion of  the  B.  coli  content  furnishes  a  good  measure  of 
the  faecal  pollution  of  water.  There  is  still  a  school  of 
bacteriologists  in  Germany,  however,  who  are  inclined 
to  place  little  value  on  the  colon-test.  We  have  pointed 
out  how  Kruse  and  his  pupils  at  Bonn  led  in  the  attack 
on  it  in  1894.  Fourteen  years  later  Kruse  (1908) 
concluded  after  a  full  summary  of  the  literature  that 
the  colon  test  was  on  the  whole  less  valuable  than  the 
gelatin  count,  although  he  admitted  that  when  the 
test  is  made  quantitative  it  is  valuable  as  a  supple- 
ment to  the  plate  count.  Konrich  (1910),  working  in 
Gartner's  laboratory  at  Jena,  after  perhaps  the  most 
exhaustive  study  ever  made  of  the  whole  subject, 
concludes,  that  to  include  the  colon  test  in  forming 
judgment  on  the  sanitary  quality  of  a  water  is  to 
complicate  the  procedure  without  improving  it  and  that 
one  would  do  well  to  omit  the  test  except  in  certain 
special  cases. 

The  German  criticisms  of  the  colon  test  are  based 


170       ELEMENTS  OF  WATER  BACTERIOLOGY 

mainly  on  two  considerations,  the  inaccuracy  of  the 
method  itself  and  the  difficulties  in  its  interpretation. 
They  hold  on  the  one  hand  that  the  errors  in  the  enrich- 
ment process  and  the  consequent  lack  of  correspondence 
between  duplicate  determinations  are  so  great  that  the 
whole  process  is  worthless.  It  is  of  course  true  that 
chance  errors  of  distribution  and  the  overgrowth  which 
often  occurs  in  the  lower  tubes  of  a  dilution  series  do 
occasionally  lead  to  individual  erroneous  results.  If 
several  dilutions  are  made  with  duplicates  in  each 
dilution,  and  particularly  if  reliance  is  placed,  as  it 
should  be  placed,  not  on  single  determinations,  but 
on  the  average  of  several  tests,  results  are,  however, 
obtained  which  are  in  accord  with  each  other  and  with 
the  results  of  practical  epidemiological  experience. 

The  other  objection  brought  forward  by  many  Ger- 
man sanitarians  is  that  the  wide  distribution  of  the  colon 
bacillus  leads  to  its  presence  even  in  considerable  num- 
bers in  waters  which  are  really  of  good  sanitary  quality. 
It  is  undoubtedly  true  that  colon  bacilli  are  often 
found  in  surface  waters  which  receive  no  sewage  but 
which  are  polluted  only  with  the  wash  from  roadways 
or  cultivated  land.  Even  dust  blowing  in  from  a  road- 
way may  perhaps  contribute  an  appreciable  pollution, 
as  we  have  pointed  out  above.  Gartner  (19 10)  cal- 
culates that  in  the  soil  of  a  cultivated  field  100 
meters  square  there  are  15,000,000,000  colon  bacilli 
and  points  out  that  it  is  no  wonder  that  a  rain  should 
wash  a  few  of  them  through  into  neighboring  wells. 
Konrich  (1910)  goes  so  far  as  to  say  that  to  reject  on 
principle  all  water  containing  B.  coli  in  i  c.c.  portions 


SIGNIFICANCE  OF  COLON  GROUP  IN  WATER     171 

is  impossible,  since  many  regions  have  no  other  water 
available. 

On  the  other  hand,  it  may  be  urged  that  it  is  better 
to  be  on  the  safe  side  if  possible.  There  is  no  doubt 
that  in  the  United  States  there  is  no  difficulty  in  secur- 
ing water  supplies,  either  from  the  ground  or  by  storage 
or  filtration,  which  only  rarely  contain  colon  bacilli 
in  i  c.c.  samples.  It  may,  perhaps,  be  that  in  the  thickly 
settled  and  intensively  cultivated  parts  of  Germany 
this  is  not  the  case.  Where  it  is  possible  to  obtain  such 
waters  we  believe  it  to  be  wise  to  do  so.  Ground 
waters  to  which  the  colon  bacilli  from  cultivated  soil 
penetrate  and  surface  waters  which  still  contain  many 
colon  bacilli  from  fields  and  roadways  may  be  generally 
inocuous  but  there  is  always  the  chance  that  infectious 
material  may  find  its  way  to  the  soil  and  may  enter, 
along  with  the  intrinsically  harmless  colon  bacilli  of 
manure.  The  two  most  recent  German  contributions 
to  the  subject  are  on  the  whole  favorable  to  the  colon 
test  if  wisely  and  intelligently  applied.  Fromme 
(1910),  from  Dunbar's  laboratory  at  Hamburg,  concludes 
from  a  survey  of  the  literature  and  from  his  own  studies 
that  "  the  finding  of  colon  bacilli  in  water  is  a  valuable 
indicator  of  the  quality  of  the  water  "  and  recommends 
it  particularly  for  ground-water  spring  waters  and 
filter  effluents;  and  Prof.  Gartner,  the  head  of  the  Hygiene 
laboratory  at  Jena,  after  a  thorough  discussion  of 
previous  work,  comes  to  a  much  more  conservative 
conclusion  than  his  pupil,  Konrich  (Gartner,  1910). 
While  emphasizing  the  shortcomings  of  the  process 
and  deploring  the  tendency  "to  set  B.  coli  on  a  high 


172       ELEMENTS  OF  WATER  BACTERIOLOGY 

throne  and  dance  before  it  "  he  recognizes  that  the 
isolation  of  this  organism  has  its  place.  "  My  aim  will 
have  been  realized,"  he  says,  "  if  I  have  been  able  to 
show  that  the  colon  test  may  be  useful  under  certain 
circumstances,  but  that  it  must  be  viewed  with  great 
caution  and  that,  moreover,  not  the  mere  numbers 
of  colon  bacilli,  but  a  careful  consideration  of  the  local 
situation  and  all  the  circumstances  bearing  on  the  special 
case  are  absolutely  essential  to  the  formation  of  an 
opinion."  With  Prof.  Gartner's  emphasis  on  the 
importance  of  a  sanitary  inspection  all  experienced 
bacteriologists  will  certainly  find  themselves  in  agree- 
ment. 

Some  of  the  best  French  bacteriologists  are  strong 
supporters  of  the  value  of  B.  coli  as  an  indicator  of 
pollution.  Gautie  (1905)  holds  that  the  quantitative 
determination  of  B.  coli  is  of  the  highest  importance 
in  water  analysis;  and  Vincent  (1905),  in  an  excellent 
review  of  the  subject,  gives  strong  reasons  for  main- 
taining the  same  position.  He  finds  B.  coli  absent 
from  spring  and  well-waters  of  good  quality  and  present 
in  polluted  water  in  proportion  to  its  pollution.  He 
concludes  finally  that  water  containing  B.  coli  in  .1  to 
i.o  c.c.  is  unfit  to  drink,  while  if  the  organism  is  found 
in  i.o  to  10.0  c.c.  it  is  of  doubtful  quality. 

In  Portugal  too  the  trend  of  opinion  is  strongly  in 
favor  of  the  colon  test — the  Anglo-American  procedure, 
as  it  is  called  in  the  publications  of  Dr.  Bettencourt 
and  his  associates  (Ferreira,  Horta  and  Paredes,  i9o8a). 

Altogether  the  evidence  is  quite  conclusive  that  the 
absence  of  B.  coli  demonstrates  the  harmlessness  of  a 


SIGNIFICANCE  OF  COLON  GROUP  IN  WATER     173 

water  as  far  as  bacteriology  can  prove  it.  That  when 
present,  its  numbers  form  a  reasonably  close  index  of 
the  amount  of  pollution,  the  authors  above  quoted  have 
proved  beyond  reasonable  cavil.  It  may  safely  be 
said  that  when  the  colon  bacillus  is  found  in  such  abun- 
dance as  to  be  isolated  in  a  large  proportion  of  cases 
from  i  c.c.  of  water,  it  is  reasonable  proof  of  the  presence 
of  serious  pollution. 


CHAPTER  VIII 

VARIETIES    OF    COLON    BACILLI  AND   THEIR   SPECIAL 
SIGNIFICANCE 

Tests  Used  for  Subdividing  the  Colon  Group.    The 

group  of  colon  bacilli  as  denned  in  Chapter  VI  includes 
all  aerobic  Gram-negative  bacilli  which  produce  acid 
and  gas  in  dextrose  and  lactose  media.  By  the  applica- 
tion of  various  tests,  mainly  bio-chemical,  it  may  be 
further  split  up,  almost  at  will,  into  a  great  number  of 
varieties.  The  principal  tests  which  have  been  used  for 
this  purpose  are  as  follows : 

1.  Motility. 

2.  Coagulation  of  milk. 

3.  Production  of  indol. 

4.  Liquefaction  of  gelatin. 

5.  Reduction  of  nitrates. 

6.  Reduction  of  neutral  red. 

7.  Fermentation  of  various  carbohydrates  other  than 
dextrose  and  lactose. 

8.  Voges  and  Proskauer  reaction. 

9.  Character  of  colonies  on  various  solid  media. 

10.  Aesculin  reaction. 

11.  Growth  and  fermentation  at  46°. 

Motility  is  seldom  determined  in  actual  routine  work 
in  this  country  from  the  general  belief  that  its  demon- 

174 


VAEIETIES  OF  COLON  BACILLI  175 

stration  is  both  burdensome  and  needless.  Motility.is  a 
fluctuating  and  uncertain  property  and  one  which 
frequently  requires  repeated  preliminary  cultivations 
to  make  manifest.  Furthermore,  non-motile  colon  bacilli 
are  common  in  the  intestine  and  are  probably  as  charac- 
teristic of  pollution  as  the  motile  forms. 

McWeeney  (1904)  found  non-motile  B.  coli  abundant 
in  faeces  and  observed  cases  where  the  organisms  were 
motile  at  20°  and  not  at  37°.  He  quotes  Stocklin  as  hav- 
ing found  116  non-motile  strains  among  300  otherwise 
normal  B.  coli  from  faeces.  Evidence  that  non-motile 
bacteria,  otherwise  resembling  B.  coli,  occur  in  unpol- 
luted water  would  furnish  the  only  basis  for  requiring 
this  test  as  a  routine  procedure.  No  such  evidence  exists. 
The  great  body  of  data  which  connects  the  presence  of 
B.  coli  with  pollution  includes  all  B.  coli  whether  motile 
or  not,  since  scarcely  any  bacteriologists  observe  this 
property  in  actual  practice. 

Howe  (1912)  has  recently  come  to  the  conclusion  that 
motility  has  no  diagnostic  value.  MacConkey,  however 
(MacConkey,  1909),  after  carefully  reviewing  the  various 
characters  suggested  for  use  in  studying  colon  bacilli, 
retains  this  one  as  important.  He  recommends  that  it  be 
made  in  a  drop  of  a  6-hour  broth  culture  on  an  ordinary 
slide  with  a  J-inch  objective  and  dark  ground  illumina- 
tion. Failure  to  show  motility  indicates  in  particular 
B.  lactis-aerogenes  and  B.  pneumonias  of  his  classifica- 
tion (see  p.  191). 

Coagulation  of  milk  is  one  of  the  most  generally  ac- 
cepted tests  for  the  colon  group;  and  as  a  rule  most 
lactose  fermenting  forms  give  a  positive  reaction.  The 


176       ELEMENTS  OF  WATER  BACTERIOLOGY 

common  practice  in  this  country  is  to  incubate  litmus 
milk  tubes  for  48  hours  at  37°  and  then  heat  to  boiling. 
Tubes  which  have  not  coagulated  spontaneously  fre- 
quently do  so  on  heating.  Biffi  (1906)  has  pointed  out 
that  milk  to  be  used  for  this  purpose  should  not  be 
sterilized  in  the  autoclave.  Temperatures  above  100° 
so  alter  the  milk  as  to  make  its  coagulation  much  slower. 
Konrich  (1910)  concluded  from  his  experiments  that 
the  coagulation  of  milk  by  the  colon  bacillus  is  often  not 
due  to  acid  production,  but  to  the  secretion  of  a  specific 
coagulating  ferment,  since  he  found  that  the  addition  of 
an  amount  of  acid  similar  to  that  produced  by  the 
organism  failed  to  coagulate. 

The  production  of  indol  in  a  peptone  solution  is  an- 
other test  very  generally  used  in  this  country  and  in 
England  as  diagnostic  of  "  typical  "  B.  coli.  The 
usual  procedure  has  been  to  incubate  a  tube  of  an 
aqueous  solution  containing  i  per  cent  peptone  and 
.01  per  cent  sodium  nitrite  for  four  days  at  37°  and  to 
test  for  indol  by  adding  i  c.c.  of  a  .02  per  cent  solution 
of  sodium  or  potassium  nitrite  and  i  c.c.  of  a  i  to  i 
solution  of  sulphuric  acid.  Both  the  tube  and  the 
reagents  should  be  cooled  on  ice  before  mixing,  and  the 
tube  should  be  left  in  a  cool  place  for  an  hour  afterward 
to  allow  time  for  the  characteristic  rose-red  color  of 
nitroso-indol  to  develop. 

Marshall  (1907)  and  other  German  and  English  work- 
ers have  shown  that  this  nitrite  and  sulphuric  acid  test 
for  indol  often  gives  incorrect  results  and  that  the  test 
modified  by  Bohme  from  Ehrlich  is  both  more  sensitive 
and  more  accurate.  Two  solutions  are  used;  No.  i  is 


VARIETIES  OF  COLON  BACILLI  177 

made  up  of  4  parts  of  para-dimethyl-amido-benzalde- 
hyde,  380  parts  of  absolute  alcohol,  and  80  parts  of  con- 
centrated HC1;  No.  2  is  a  saturated  aqueous  solution 
of  potassium  persulphate ;  5  c.c.  of  i  is  added  to  10  c.c. 
of  a  broth  culture  and  then  5  c.c.  of  2  is  added  and  the 
whole  shaken.  A  red  color  indicates  indol.  Mac- 
Conkey  (1909)  who  was  at  first  inclined  to  discard  indol 
as  a  routine  test,  believes  that  when  made  in  this  way 
it  is  of  much  value. 

Howe  (1912),  from  a  statistical  study  of  630  strains 
of  intestinal  colon  bacilli,  concluded  that  indol,  ammonia, 
and  nitrite  tests  were  but  slightly  correlated  with  general 
vigor  and  had  but  slight  classificatory  significance.  It 
does  not  necessarily  follow,  however,  that  this  is  true 
of  the  forms  which  occur  in  stored  waters.  The  so- 
called  "  atypical  B.  coli  "  are  of  course  rare  in  faeces  but 
they  may  occur  in  sufficient  numbers  to  be  important 
in  waters  which  have  been  remotely  polluted. 

The  liquefaction  of  gelatin  is  another  test  generally 
applied  in  any  detailed  study  of  the  colon  group.  The 
longer  the  tubes  are  kept  the  higher  will  be  the  propor- 
tion of  positive  results,  for  there  are  many  slowly  lique- 
fying forms  grading  by  almost  imperceptible  degrees 
into  the  commoner  non-liquefying  type.  The  table 
cited  from  Gage  and  Phelps  (1903)  on  p.  186  shows  that 
of  a  series  of  1908  cultures  from  various  waters,  sewages, 
and  shellfish  8  per  cent  liquefied  gelatin  in  4  days,  10 
per  cent  in  7  days,  13  per  cent  in  10  days,  and  17  per 
cent  in  14  days. 

The  reduction  of  nitrates  to  nitrites  has  been  used  in 
the  United  States  as  one  of  the  five  standard  tests  for 


178       ELEMENTS  OF  WATER  BACTERIOLOGY 

B.  coli,  but  it  has  never  gained  wide  acceptance  in  Eng- 
land or  Germany.  The  usual  practice  has  been  to 
incubate  for  4  days  at  37°  and  to  test  for  nitrites  by 
adding  a  drop  of  each  of  the  following  solutions  in  suc- 
cession : 

A.  Sulphanilic  acid 5  gram 

Acetic  acid  (25%  sol.) 150.0  c.c. 

B.  Naphthylamine  chloride o.  i  gram 

Distilled  water 20.0  c.c. 

Acetic  acid  (25%  sol.) 150.0  c.c. 

A  red  or  violet  coloration  indicates  the  presence  of  nitrites. 

In  making  the  nitrite  test  it  is  important  to  remember 
the  possibility  that  appreciable  amounts  of  nitrite  may 
be  present  in  the  media — either  derived  from  the  air  or 
from  the  use  of  impure  peptone  (Wherry,  1905).  In  the 
case  of  the  nitrite  reaction  control  tubes  should  always 
be  tested  from  the  same  batch  of  media  and  only  a 
distinct  red  color  should  be  considered  positive.  The 
nitrite  test  is  particularly  subject  to  variations  of  un- 
explained origin.  Of  two  duplicate  tubes  inoculated  in 
the  same  way,  one  may  show  a  strong  reaction  and  the 
other  none. 

The  reduction  of  neutral  red  has  been  extensively  used 
in  England  and  less  in  this  country.  It  has  been  re- 
ferred to  in  Chapter  VI.  as  one  of  the  tests  suggested  for 
use  as  a  presumptive  indicator  of  the  colon  group  as  a 
whole.  MacConkey  (1909)  concludes  that  both  the  ni- 
trate test  and  the  neutral  red  test  should  be  dropped 
from  the  procedure  used  in  identifying  colon  bacilli,  since 
so  many  organisms  give  these  reactions  that  they  have 
little  significance.  Houston,  however,  in  the  important 
investigations  which  will  shortly  be  discussed,  used  the 


VARIETIES  OF  COLON  BACILLI  179 

production  of  a  greenish  fluorescence  in  neutral  red 
broth  as  one  of  his  tests  of  "  typical  "  B.  coli  and  ap- 
parently found  it  valuable. 

Fermentation  of  various  carbohydrate  media  has  be- 
come the  most  common  method  of  subdividing  the 
bacilli  of  the  colon  group  during  the  last  few  years,  largely 
as  a  result  of  the  work  of  MacConkey.  Sugar  broths  for 
this  test  are  generally  put  up  in  fermentation  tubes  of 
some  sort  so  that  the  gas  formation  may  be  observed 
and  perhaps  measured,  while  acid  production  may  be 
indicated  by  the  addition  of  litmus  or  accurately  deter- 
mined by  titration.  The  old-fashioned  fermentation 
tube  with  a  bulb  and  a  stand  has  given  way  in  most 
water  laboratories  to  a  plain  bent  tube  of  even  bore 
and,  more  recently,  to  a  still  simpler  device,  a  small  vial 
inverted  in  an  ordinary  test-tube  of  sugar  broth.  The 
air  in  the  top  of  the  vial  is  driven  out  on  sterilization 
and  the  presence  or  absence  of  gas  can  be  easily  deter: 
mined,  although  it  is  not  possible  to  measure  its  quan- 
tity with  accuracy.  If  an  ordinary  bent  tube  is  used 
the  amount  of  gas  in  the  closed  arm  may  be  conven- 
iently measured  by  the  Frost  gasometer  (Frost,  1901). 
If  a  measurement  of  the  gas  ratio  is  desired  a  few  centi- 
meters of  strong  sodium  or  potassium  hydrate  are  added 
and  mixed  with  the  broth  by  cautiously  tipping  the 
tube;  a  second  measurement  determines  the  amount  of 
gas  absorbed  (assumed  to  be  C02) . 

It  has  been  pointed  out  in  Chapter  VI  that  the  gas 
ratio  appears  to  be  a  reaction  of  slight  importance  as 
thus  determined. 

The  list  of  fermentable  substances  used  by  various 


180       ELEMENTS  OF  WATER  BACTERIOLOGY 

observers  in  classifying  colon  bacilli  is  a  long  one.  It 
was  pointed  out  by  Smith  (1893)  long  ago  that  saccharose 
divides  these  organisms  into  two  groups,  and  Winslow 
and  Walker  (1907)  have  found  that  strains  which 
attack  saccharose  generally  ferment  raffinose  also. 
MacConkey  (1909)  has  made  the  most  careful  study 
of  the  reactions  of  the  group  during  recent  years.  He 
believes  that  of  the  ordinary  tests,  milk  and  neutral 
red  should  be  discarded  and  fermentative  reactions  in 
saccharose,  dulcite,  adonite,  inulin,  inosite  and  mannite 
should  be  substituted  in  addition  to  motility,  the  indol 
test,  the  liquefaction  of  gelatin,  and  the  Voges  and 
Proskauer  reaction. 

Howe  (1912),  from  his  recent  statistical  study  of  630 
strains  of  intestinal  bacilli,  concluded  that  fermenta- 
tion tests  in  mannite,  dulcite,  and  starch  media  are 
of  little  value  in  the  classification  of  colon  bacilli,  since 
they  are  not  closely  correlated  with  other  characters. 
It  should  be  noted,  however,  that  he  worked  only  with 
fresh  intestinal  strains  and  it  is  possible  that  types 
characterized  by  definite  reactions  in  these  media  may 
be  rare  in  faeces  but  may  develop  so  as  to  be  important 
in  stored  waters. 

The  Voges  and  Proskauer  reaction  has  been  extensively 
used  by  MacConkey  and  his  followers  in  England  and 
by  Bergey  and  Deehan  (1908)  in  this  country.  After 
the  carbon  dioxide  in  the  fermentation  tube  has  been 
absorbed  by  caustic  soda,  if  the  tube  be  allowed  to  stand, 
an  eosin-like  color  gradually  develops  in  the  open 
arm,  due  to  the  presence  of  acetyl-methyl-carbinol. 
West  (1909)  points  out  that  the  test  used  by  Rivas, 


VARIETIES  OF  COLON  BACILLI  181 

the  boiling  of  1-4  c.c.  of  a  48-hour  dextrose  broth 
culture  with  5  c.c.  of  a  10  per  cent  caustic  soda  solu- 
tion, is  a  quick  method  of  obtaining  the  Voges  and 
Proskauer  reaction.  A  yellow  color  is  produced  under 
these  conditions  by  the  sugar  alone,  and  a  pinkish 
eosin-like  color  when  the  acetyl-methyl-carbinol  is 
present.  The  reaction  is  hastened  by  shaking  or 
blowing  into  the  tube  to  promote  oxidation.  West 
confirms  the  conclusion  of  MacConkey  and  Bergey 
that  this  reaction  is  characteristic  of  the  B.  lactis- 
aerogenes  and  B.  cloacae  types  (saccharose  positive, 
dulcite  negative  organisms). 

The  characters  of  colonies  on  various  solid  media, 
such  as  Endo  agar  or  Conradi-Drigalski  agar  appear 
to  be  of  minor  importance,  usually  depending  on  one 
of  the  simple  fermentative  reactions  for  their  differential 
value.  The  aesculin  test  and  the  Eijkman  test  (fer- 
mentation at  46°)  have  been  discussed  in  connection 
with  their  use  as  enrichment  procedures  in  Chapter  VI. 

Biological  Significance  of  Variations  in  the  Colon 
Group.  The  general  view  among  water  bacteriologists 
has  been  that  forms  differing  from  the  "  typical  "  B. 
coli  in  one  or  more  respects  represented  original  intes- 
tinal types  weakened  by  a  prolonged  sojourn  in  an 
unfavorable  environment.  As  Whipple  says  (Whipple, 
1903),  "  The  type  form  of  Bacillus  coli  is  one  which 
can  be  defined  within  reasonably  narrow  limits,  but 
when  the  organism  has  been  away  from  its  natural 
habitat  for  varying  periods  of  time,  and  has  existed 
under  abnormal  conditions,  its  ability  to  react  nor- 
mally to  the  usual  tests  appears  to  be  greatly  im- 


182       ELEMENTS  OF  WATER  BACTERIOLOGY 

paired.  Its  power  to  reduce  nitrates  may  be  lost,  or 
on  the  other  hand,  may  be  increased;  its  power  to 
produce  indol  may  be  lost,  or  on  the  other  hand,  it 
may  be  increased;  its  power  to  coagulate  milk,  even, 
is  sometimes  reduced,  although  seldom  entirely  lost; 
its  power  to  ferment  carbohydrates  may  be  altered 
so  that  the  amount  of  gas  obtained  in  a  fermentation- 
tube,  as  well  as  its  ratio  of  H  to  C02,  is  quite  abnormal. 
But  in  spite  of  all  these  facts,  the  bacillus  tested  may 
have  been  originally  a  true  Bacillus  coli." 

The  results  obtained  by  Peckham  (1897)  suggest 
that  the  indol  reaction  in  particular  is  highly  variable. 
By  successive  daily  transfers  in  peptone  broth  she  was 
able  to  increase  the  amount  of  indol  produced  by  nor- 
mal B.  coli,  and  by  a  longer  continuance  of  the  same 
process  to  again  weaken  and  abolish  the  power  of  form- 
ing it.  Gas  formation  too  was  slackened  in  the  cul- 
tures grown  for  too  many  transfers  in  the  same  medium. 
Horrocks  (1903)  found  that  B.  coli  kept  in  unsterilized 
well-waters  and  tap  waters  and  in  sterilized  sewage  and 
Thames  water  for  2  to  3  months  showed  only  a  feeble 
indol  production  and  a  delayed  action  on  milk  and 
neutral  red.  These  modified  forms  are  sometimes 
called  "  atypical  B.  coli,"  or  "  para-colon  bacilli,"  and 
Vincent  gives  them  the  picturesque  name,  "  microbic 
satellites  of  B.  coli." 

'  Such  anomalies  are  most  frequent  with  cultures 
freshly  isolated  from  water,  and  they  may  often  be 
avoided,  as  Fuller  and  Johnson  (1899)  have  shown, 
by  subjecting  the  organism  to  a  process  of  preliminary 
cultivation.  For  this  purpose  the  American  Public 


VARIETIES  OF  COLON  BACILLI  183 

Health  Association  Committee  recommends  three  suc- 
cessive cultivations  in  broth  at  20  degrees,  each  of  24 
hours'  duration,  inoculation  from  the  last  broth  tube 
of  a  gelatin  plate  which  is  incubated  for  48  hours  at  20 
degrees,  inoculation  of  an  agar  streak  from  one  colony 
on  the  plate  and  incubation  of  this  streak  for  48  hours 
at  20  degrees. 

Often,  however,  the  differences  between  types  of  the 
colon  group  indicate  something  much  more  funda- 
mental than  temporary  weakening  due  to  unfavorable 
environment.  In  particular  sudden  more  or  less  perma- 
nent mutations  may  suddenly  appear.  Twort  (1907) 
reports  that  by  continued  cultivation  in  sugar  media  he 
was  able  to  develop  fermentative  power  in  certain  mem- 
bers of  the  Gartner  group  which  lacked  such  powers 
before. 

The  work  of  Massini,  Miiller,  Sauerbeck,  Konrich 
and  others  (well  summarized  by  Konrich,  1910)  has 
also  shown  that  mutations  capable  of  fermenting  sugars 
may  suddenly  arise  from  a  parent  strain  lacking  this 
power.  Burri  (1910)  has  contributed  to  the  same 
problem  and  has  found  that  the  latent  power  to  ferment 
a  given  sugar  is  released  by  growing  the  organism  on 
that  particular  sugar,  but  that  as  Konrich  and  the  others 
show  only  a  certain  proportion  of  the  cells  develop 
this  power.  The  most  important  recent  studies  of 
bacterial  mutation  have  been  made  by  Penfold.  In 
his  latest  communication  (Penfold,  1912)  he  shows 
that  many  bacteria  of  the  colon-typhoid  group  pro- 
duce a  mutant  capable  of  fermenting  lactose,  that 
all  strains  of  the  typhoid  bacillus  produce  dulcite  and 


184       ELEMENTS  OF  WATER  BACTERIOLOGY 

iso-dulcite  mutants,  that  many  paratyphoid  and  Gartner 
group  bacilli  produce  raffinose  mutants,  and  that  other 
mutations  also  occur.  The  general  phenomena  are 
the  same  in  each  case.  A  strain  which  normally  fails 
to  ferment  a  given  carbohydrate  is  grown  upon  a  solid 
medium  containing  that  carbohydrate.  As  the  colonies 
develop  there  appear  upon  them  raised  papillae  of  a 
different  consistency  from  the  rest  of  the  colony  and 
colored  red  if  litmus  be  present.  Transplants  from 
the  papillae  give  pure  cultures  of  a  strain  fermenting 
the  carbohydrate  in  question  and  forming  no  papillae. 
Transplants  from  the  other  portions  of  the  colony 
give  the  original  strain,  non-fermenting,  but  capable 
of  producing  fermenting  mutants  as  before.  The 
identity  of  derivative  strain  in  all  other  respects  has 
been  made  clear  by  exhaustive  cultural  tests  and  serum 
reactions ;  and  Penf old  has  shown  that  the  whole  proc- 
ess may  be  repeated,  starting  from  an  isolated  single 
cell. 

The  work  of  MacConkey  and  Clemesha,  which  will 
be  discussed  shortly,  is  based  on  the  assumption  that 
a  great  number  of  minute  subdivisions  of  the  colon 
group,  whether  they  have  arisen  by  the  gradual  modify- 
ing effect  of  an  unfavorable  environment,  or  by  muta- 
tions, or  in  some  other  way,  are  for  practical  purposes 
fairly  permanent  entities  which  they  describe  and  name 
as  definite  species. 

Distribution  of  Types  of  the  Colon  Group  in  Waters 
of  Various  Kinds.  The  sanitary  importance  of  a  study 
of  these  minor  types  within  the  colon  group  depends 
on  the  assumption  that  a  certain  set  of  characters  is 


VARIETIES  OF  COLON  BACILLI  185 

generally  associated  with  forms  fresh  from  the  intes- 
tine and  may  therefore  be  called  typical.  Such  "  typ- 
ical" B.  coli  are  understood  as  a  rule  to  be  motile, 
to  clot  milk,  produce  indol,  reduce  nitrate  and  neutral 
red  and  to  fail  to  liquefy  gelatin.  It  seems  clear  that 
forms  having  these  characters  predominate  in  the 
intestine  itself  while  differing  or  "  atypical"  forms 
bear  to  them  somewhat  the  relation  implied  in  Vincent's 
term,  satellites.  Houston  (1903*)  examined  in  detail 
10 1  cultures  of  coli-like  microbes  isolated  from  faeces  and 
found  that  72  per  cent  of  the  cultures  were  typical  in 
all  respects,  while  n  per  cent  more  differed  only  in 
being  non-motile.  The  remaining  17  per  cent  were 
atypical,  reacting  abnormally  to  milk,  indol,  neutral 
red,  litmus  whey  or  Capaldi  and  Proskauer's  medium. 
In  a  later  investigation,  Houston  (1904)  made  a  careful 
study  of  the  distribution  of  the  atypical  forms  in  faeces, 
sewage,  polluted  water,  and  the  filtered  water-supplies 
of  London.  According  to  his  ingenious  system  of 
nomenclature,  "  fl  "  indicates  an  organism  which  pro- 
duces green  fluorescence  in  neutral  red  broth;  "  ag," 
one  which  forms  acid  and  gas  in  lactose  media;  "  in," 
one  which  produces  indol;  and  "  ac  "  one  which  acidifies 
and  clots  litmus  milk.  The  combination  of  all  these 
properties  gives  "  Flaginac,"  or  typical  B.  coli;  "  aginac" 
is  a  form  which  fails  to  reduce  neutral  red;  "  flagac," 
one  which  fails  to  form  indol,  etc.  "  Flaginac  "  B.  coli 
form  the  great  majority  of  coli-like  microbes  in  faeces, 
but  Houston  found  that  in  filtered  water  they  are 
outnumbered  by  atypical  forms,  of  which  he  recognized 
thirty-five  distinct  types. 


186       ELEMENTS  OF  WATER  BACTERIOLOGY 


PERCENTAGE  OF  CULTURES  PASSING  VARIOUS  TESTS  IN  THE  ROUTINE  EXAMINATION  FOR 
B.  COLI  AT  THE  LAWRENCE  EXPERIMENT  STATION  OF  THE  MASSACHUSETTS  STATE  BOARD 
OF  HEALTH  (GAGE  AND  PHELPS,  1903) 

•ii°o  'a 

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vo  vo  vo   rt*    T^-    ••^- 

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*Including  cultures  which  failed  to  grow  on  agar  and  streptococcus  cultures,  giving  a  very  scanty,  non-characteristic  growth. 

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VAEIETIES  OF  COLON  BACILLI  187 

On  the  whole,  much  of  the  English  evidence  tends  to 
the  assumption  that  the  atypical  forms,  or  "  paracolon 
bacilli,"  generally  represent  weakened  strains  from  the 
intestinal  B.  coli  stock.  As  Savage  says,  "  we  know 
that  nearly  all  the  coli-like  organisms  in  faeces  are  quite 
typical  B.  coli,  that  in  sewage  a  good  many  atypical 
varieties  are  present,  and  that  in  contaminated  water 
and  soil  the  proportion  present  is  still  larger." 

The  data  tabulated  on  p.  186  from  Gage  and  Phelps 
(1903)  lead  to  a  similar  conclusion.  About  60  per  cent 
of  the  cultures  isolated  from  polluted  river  water, 
filtered  water,  and  sewage  proved  to  be  typical  B.  coli, 
while  41  and  43  per  cent  of  those  isolated  from  spring 
water  and  shellfish,  respectively,  and  48  per  cent  of 
those  from  ice  belonged  in  this  class. 

Contradictory  results,  indicating  a  higher  proportion 
of  typical  forms  outside  the  body  than  within  it,  have 
been  obtained  by  Konrich  (1910)  in  the  examination  of 
2387  different  strains  isolated  in  about  equal  propor- 
tions from  faeces,  earth,  and  water.  Of  the  2387  coli- 
like  microbes  studied,  308  were  excluded  by  micro- 
scopic examination  (showing  abnormal  morphology  or 
positive  Gram  reaction)  or  by  their  liquefaction  of 
gelatin.  The  other  2079  strains  were  tested  in  sugar 
media  and  peptone  water  with  the  results  tabulated 
on  p.  188. 

We  are  inclined  to  attribute  these  results  of  Konrich's 
largely  to  the  technique  which  he  employed.  It  seems 
to  be  clearly  stated  in  his  paper  that  he  obtained  his 
faecal  cultures  by  direct  plating  on  solid  media,  while 
his  earth  and  water  samples  were  treated  to  preliminary 


188       ELEMENTS  OF  WATER  BACTERIOLOGY 


BIOCHEMICAL  REACTIONS  OF  2079  ORGANISMS  OF    THE 
COLON   GROUP 

(KONRICH,  1910) 


Medium. 

Reaction. 

Percentage  of  Positive 
Results. 

Faecal 
Strains. 

Earth 
Strains. 

Water 
Strains. 

Dextrose  broth  
Dextrose  broth,  46°.  .  .  . 
Lactose  broth  
Lactose  broth. 

Gas  production  
Gas  production  
Gas  production  
Acid  production  .... 
Coagulation  

IOO 

59 

77 
97 
65 

54 
38 

60 

IOO 

82 

87 
IOO 

84 

68 
57 

77 

IOO 

82 
92 
IOO 

80 

65 

57 

76 

Milk  
Neutral    red    dextrose 
agar  
Peptone  solution  
Endo  agar. 

Fluorescence 

Indol  production.  .  .  . 
Deep    red    colonies 
with  greenish  luster 

enrichment  in  sugar  broths.  Such  an  enrichment  would 
undoubtedly  tend  to  increase  the  proportion  of  typical 
B.  coli.  Konrich's  own  experiments  on  the  storage 
of  pure  cultures  of  colon  bacilli  in  water  showed  a  gen- 
eral, though  not  invariable,  relative  increase  of  atypical 
forms  as  the  sojourn  in  water  became  long  continued. 
Houston  (1911)  has  recently  pointed  out  the  danger 
that  the  selective  action  of  enrichment  media  may 
confuse  the  normal  relations  of  the  bacterial  flora; 
and  in  order  to  obtain  a  more  accurate  idea  of  the 
relations  involved  he  made  an  elaborate  study  of  raw 
water,  stored  water,  and  stored  and  filtered  water  by 
direct  plating,  preceded  by  the  use  of  various  physical 
concentration  methods.  The  results  indicated  in  the 
table  on  p.  189  are  of  interest. 


VARIETIES  OF  COLON  BACILLI 


189 


CHARACTERISTICS    OF    DEXTROSE    FERMENTING     BAC- 
TERIA FROM  RAW,  STORED,  AND  FILTERED  WATERS 

(HOUSTON,  19  n) 


Per  Cent  Positive  Results. 

,  « 

a 

Gas  Production. 

en  O 

•g 

n*  " 

Source. 

w 

§ 

A 

c 

. 

a 

6 

i 

JQ 
U 

%* 

1 

1§ 

1 

1 

"3 
a 
i—  i 

o 
a 

|§ 

Raw          river 

water   . 

320 

76 

40 

2Z 

C7 

44 

I  c; 

7 

O    3 

63 

T  r 

Stored  water.  . 

232 

57 

40 

31 

35 

25 

6 

0.4 

66 

6 

Stored  and  fil- 

tered water  . 

225 

69 

24 

34 

Si 

25 

5 

3 

40 

6 

The  strains  having  the  combination  of  positive  reac- 
tions in  lactose  and  peptone  solution  made  up  53  per  cent 
of  those  isolated  from  the  raw  waters.  46  per  cent  of 
those  from  the  stored  waters,  and  34  per  cent  of  those 
from  the  stored  and  filtered  waters. 

MacConkey's  Classification  of  the  Colon  Group. 
MacConkey  is  not  satisfied  with  this  general  classifica- 
tion of  colon  bacilli  into  typical  and  atypical  forms, 
but  wishes  to  go  much  further,  believing  that  even  the 
so-called  "  typical  B.  coli  "  chould  rather  be  con- 
sidered a  complex  including  a  considerable  number  of 
definite  individual  types.  After  a  detailed  study  of  the 
reactions  of  the  lactose-fermenting  bacteria  of  faeces 
he  outlined  a  new  classification  based  on  fermentative 
reactions  in  the  rarer  sugars  (MacConkey,  1905). 
Using  saccharose  and  dulcite  he  first  divided  the  lactose- 
fermenting  organisms  into  four  groups  as  indicated  in 
the  table  on  p.  190. 


190       ELEMENTS  OF  WATER  BACTERIOLOGY 
PRIMARY  SUBDIVISIONS  OF  THE   COLON   GROUP 


Saccharose. 

Dulcite. 

Type. 

I. 

I 

B.  acidi-lactici 
B.  coli  (or  B.  communis) 
B.  neapolitanus  (or  B.  communior) 
B.  aerogenes 

The  fourth  group,  fermenting  saccharose,  but  not 
dulcite,  was  further  subdivided  by  MacConkey  into 
the  B.  coscoroba  type,  which  does  not  liquefy  gela- 
tin or  give  the  Voges  and  Proskauer  reaction,  the  B. 
lactis-aerogenes  type,  which  does  not  liquefy  gelatin, 
but  does  give  the  Voges  and  Proskauer  reaction,  and  the 
B.  cloacae  type,  which  liquefies  gelatin  and  gives  the 
Voges  and  Proskauer  reaction.  Records  of  the  preva- 
lence of  the  four  principal  groups  in  human  and  animal 
faeces  and  in  milk  are  given  in  MacConkey's  two  papers 
(1905  and  1909)  as  well  as  their  relative  numbers  in  a 
suspension  of  faeces  in  water  after  various  intervals  of 
time.  The  results  do  not,  however,  appear  to  us  to 
justify  any  important  practical  conclusions. 

In  his  later  paper  MacConkey  (1909)  carried  the 
sub-division  of  the  colon  group  much  further.  He 
isolated  497  lactose-fermenting  bacilli  from  the  faeces 
of  man  and  animals,  from  sewage,  water,  grains,  etc. 
All  were  Gram-negative,  fermented  lactose,  coagulated 
milk  and  reduced  nitrate.  They  were  subdivided 
by  their  action  on  gelatin,  pepton  and  various  fer- 
mentable substances  and  by  their  motility  into  over 
100  types  of  which  the  more  important  have  received 


VARIETIES  OF  COLON  BACILLJ 


191 


names.     The  principal  types  of  this  classification  are 
indicated  in  the  table  below: 

MAcCONKEY'S  CLASSIFICATION  OF  THE  COLON  GROUP 

(MACCONKEY, 


Group. 

No. 

Name. 

Liquefaction  of 
Gelatin. 

s 

Indol  Produc- 
tion. 

Fermentation  of 

J| 
II 

Saccharose. 

Dulcite. 

Adonit. 

Inulin. 

1 

I. 

i 

2 

3 

4 
5 

I 

+ 

; 

- 

- 

1 

I 

- 

\ 

B.  acidi-lactici  
B.  levans 

B.  Griinthal  
B.  vesiculosus  

II.     !  34 
35 

B.  coli  communis  
B.  Schafferi  

- 

; 

: 

t 

T 

- 

t 

i 

i 

III. 

65 

68 

7i 

72 

B.  oxytocus  pernicio- 

sus 

+ 

| 

I 

B.  pneumonias  

B.  neapolitanus  

IV. 

103 
104 
107 
108 

B.  lactis  aerogenes.  .  . 
B.  gasoformans 

+ 

; 

i 

+ 

•r- 

1 

- 

it 

=fc 

B.  coscoroba  

B.  cloacae 

In  this  country  the  MacConkey  classification  was 
first  adopted  by  Bergey  and  Deehan  (1908).  These 
workers  used  8  diagnostic  characters,  motility,  indol 
production,  liquefaction  of  gelatin,  the  Voges-Pros- 
kauer  reaction,  and  the  fermentation  of  saccharose, 
dulcite,  adonite  and  inulin.  They  tabulated  256  different 
combinations  of  these  8  characters  and  in  the  examina- 


192       ELEMENTS  OF  WATER  BACTERIOLOGY 

tion  of  92  colon-like  bacilli  from  50  samples  of  milk, 
8  of  sewage  and  i  of  kefir  they  found  43  of  the  possible 
combinations. 

Copeland  and  Hoover  (1911)  have  recently  urged 
the  importance  of  these  fermentative  reactions  in  the 
rarer  carbohydrates  in  the  study  of  the  colon  group. 
They  confirm  the  positive  Voges  and  Proskauer  reaction 
reported  by  other  observers  for  B.  lactis-aerogenes  and 
B.  cloacae  and  point  out  that  B.  lactis-aerogenes  is 
the  only  form  in  a  considerable  series  studied  which 
gives  a  brown  coloration  in  aesculin  media  in  one  day. 
On  the  other  hand  they  record  a  positive  dulcite  reac- 
tion for  B.  lactis-aerogenes  and  B.  cloaca3  which  is 
highly  confusing  and  makes  it  difficult  to  interpret 
their  results.  Both  these  names  according  to  the  usage 
of  MacConkey,  which  has  been  accepted  for  the  past 
five  years,  are  applied  to  dulcite-negative  saccharose- 
positive  organisms. 

Still  another  classification  of  the  colon  group  is  Jack- 
son's modification  of  MacConkey's  scheme  in  which 
MacConkey's  four  primary  groups  are  symmetrically 
subdivided  according  to  reactions  in  mannite  and  rafn- 
nose  with  motility,  indol  production,  nitrate  reduc- 
tion, liquefaction  of  gelatin  and  coagulation  of  milk  as 
secondary  differential  characters  (Jackson,  1911).  Under 
each  of  the  four  groups,  B.  communior  (MacConkey's 
B.  neapolitanus) ,  B.  communis  (MacConkey's  B.  coli), 
B.  aerogenes  (MacConkey's  Group  IV),  and  B.  acidi- 
lactici,  he  distinguishes  four  types,  A  (fermenting 
both  mannite  and  raffinose,  B  (mannite +,  raffinose  — ), 
C  (mannite  —  ,  raffinose  -}-),  and  D  (fermenting  neither 


VARIETIES  OF  COLON  BACILLI 


193 


mannite  nor  raffinose);  and  he  indicates  reactions  in 
other  media  by  subscript  letters.  These  types  with 
their  subtypes  are  fully  discussed  in  the  last  report 
of  the  Committee  on  Standard  Methods  of  Water 
Analysis  (1912). 

Clemesha's  Investigation  of  Stored  Waters  in  India. 
The  most  suggestive  contribution  to  this  subject  which 
has  been  made  in  recent  years  is  a  book  by  Major  W. 
W.  Clemesha  of  the  Indian  Medical  Service  on  The 
Bacteriology  of  Surface  Waters  in  the  Tropics  (Clemesha, 
1912*),  in  which  a  vigorous  argument  is  made  for  the 
MacConkey  classification  in  practical  water  work. 
Major  Clemesha's  researches  show  the  prevalence 
of  considerable  numbers  of  all  of  MacConkey's  primary 
types  in  human  and  bovine  fasces  as  indicated  in  the 
table  below,  although  the  relative  proportions  found 
in  England  and  in  India  do  not  correspond  very  closely. 
Clemesha's  percentages  are  of  special  importance 
because  they  are  based  in  each  case  on  over  1000 
colonies. 

RELATIVE     PROPORTION    OF    MACCONKEY'S    GROUPS    IN 
HUMAN    F/ECES    AND    IN    COW    DUNG 


Human  Fasces. 

Cow  Dung. 

Group. 

MacConkey. 

Clemesha. 

MacConkey. 

Clemesha.  * 

I 

34 

53 

17 

40 

2 

38 

17 

25 

9 

3 

15 

7 

48 

16 

4 

12 

22 

12 

35 

Both  in  human  faeces  and  in  cow   dung   Clemesha 
finds  the  prevailing  types  to  be  B.  coli,  B.  Grim  thai, 


194      ELEMENTS  OF  WATER  BACTERIOLOGY 

and  B.  coscoroba,  the  three  together  usually  making 
up  75  per  cent  all  the  lactose-fermenting  organisms 
present.  A  very  interesting  point  brought  out  in  these 
investigations  was  the  occurrence  of  "  epidemics," 
of  particular  types  which  at  certain  periods  become 
suddenly  frequent,  usually  prevailing  in  human  faeces, 
cow  faeces  and  water  supplies  at  the  same  time.  (It 
should  be  noted  for  the  benefit  of  anyone  studying 
Clemesha's  book  that  the  tabular  classification  of  the 
colon  group  at  the  end  contains  a  serious  misprint. 
B.  lactis-aerogenes,  B.  gasoformans,  B.  coscoroba  and 
B.  cloacae  are  there  given  as  saccharose  negative, 
whereas  they  should  be  saccharose  positive.)  The 
discussion  in  the  text,  however,  appears  to  refer  to  the 
orthodox  MacConkey  types.  Clemesha  (191 2a)  made 
a  number  of  experiments  on  the  relative  resistance  of 
the  various  lactose-fermenting  types  by  placing  faecal 
emulsions,  with  or  without  sand,  in  shallow  dishes 
in  the  sunlight  and  at  various  intervals  isolating  10 
colonies  of  the  predominant  types  and  working  out 
their  fermentative  reactions.  In  general  the  experiments 
showed  B.  coli  to  be  the  dominant  form  at  the  beginning. 
It  quickly  disappeared,  however,  and  after  a  few  hours 
B.  lactis  aerogenes,  B.  acidi-lactici,  B.  cloacae  and 
others  appeared.  At  the  end  of  the  experiments, 
often  on  the  second  day,  B.  Grim  thai  or  B.  cloacae  were 
generally  the  only  forms  surviving.  In  a  long  series  of 
examinations  of  Red  Hills  Lake  Clemesha  obtained  138 
colonies  of  lactose-fermenting  organisms  during  rainy 
periods  and  of  these  59  belonged  to  MacConkey's 
Group  I,  10  to  Group  II,  14  to  Group  III  and  55  to 


VARIETIES  OF  COLON  BACILLI 


195 


Group  IV.  Of  280  colonies  isolated  during  dry  periods, 
37  belonged  to  Group  I,  22  to  Group  III  and  221  to 
Group  IV.  When  the  forces  of  self-purification  had 
been  at  work,  Group  II  (B.  coli)  entirely  disappeared 
and  Group  IV  (B.  cloacae  and  B.  coscoroba)  was  pre- 
dominant. B.  Griinthal  was  the  commonest  of  the 
Group  I  forms.  B.  cloacae  was  especially  prevalent 
in  bottom  samples. 

A  study  of  a  number  of  rivers  in  Bengal  gave  the 
results  tabulated  below. 

RELATIVE  PREVALENCE  OF  CERTAIN  LACTOSE- 
FERMENTING  TYPES   IN   BENGAL   RIVERS 


MacConkey 
Group. 

Types. 

Dry  Weather, 
Dec.-June. 

Wet  Weather, 
July-Nov. 

I. 
II. 

IV. 

Do. 

B.  Griinthal  and  B.  vesiculosus 
B.  coii  communis  
B.  lactis  aerogenes  
B.  cloacae 

41 
3 
7 
ii 

23 
13 

19 

4 

There  are  many  irregularities  in  Dr.  Clemesha's 
results.  For  example,  B.  aerogenes,  as  well  as  the  other 
representatives  of  Group  IV,  was  more  abundant  in  Red 
Hills  Lake  during  dry  periods  than  at  times  of  rain. 
On  the  whole,  however,  it  does  seem  clear  that  his 
results  justify  a  general  classification  of  the  lactose- 
fermenting  organisms  into  three  main  groups  accord- 
ing to  resistance.  B.  coli  communis  and  B.  oxytocus 
perniciosus  (representing  MacConkey's  Groups  II  and 
III,  both  fermenting  dulcite)  are  sensitive  organisms 
found  in  numbers  only  where  pollution  is  fresh.  B.  lactis- 
aerogenes,  representing  the  subgroup  of  MacConkey's 


196       ELEMENTS  OF  WATER  BACTERIOLOGY 

Group  IV  which  ferments  adonit,  but  does  not  form 
indol  or  liquefy  gelatin,  occupies  a  somewhat  inter- 
mediate position,  appearing  in  waters  which  have  been 
fairly  recently  polluted  and  later  disappearing  again. 
Finally  B.  Grim  thai  and  B.  vesiculosus  (MacConkey's 
Group  I,  negative  in  both  saccharose  and  dulcite) 
and  B.  cloacae  and  B.  coscoroba  (of  MacConkey's 
Group  IV,  dulcite  negative  and  saccharose  positive), 
are  highly  resistant  organisms  which  occur  in  relatively 
high  proportions  in  stored  waters.  B.  cloacae  is  most 
abundant  in  bottom  sediments  and  B.  Griinthal  and  B. 
vesiculosus  in  sunned  surface-waters. 

The  moral  drawn  by  Major  Clemesha  is  that  for 
Indian  conditions  with  waters  stored  in  warm  sunned 
lakes  and  large  rivers,  where  sensitive  faecal  bacteria 
have  ample  opportunity  to  die  out  and  resistant  faecal 
bacteria  have  an  ample  opportunity  to  multiply,  it  is 
not  proper  to  condemn  water  containing  any  members 
of  the  colon  group  without  distinguishing  between  the 
more  and  the  less  resistant  forms.  For  example,  he 
quotes  239  examinations  of  which  only  74  showed  no 
B.  coli  according  to  the  English  standard,  which  closely 
corresponds  to  our  own,  while  165  showed  what  we 
should  call  positive  results.  Of  the  165,  however,  69 
contained  only  the  highly  resistant  B.  Griinthal  and 
59  contained  mixtures  of  other  forms  not  belonging 
to  MacConkey's  Group  II  (saccharose  negative,  dulcite 
positive).  Thus  of  the  239  samples  31  per  cent  would 
have  been  passed  by  Houston's  standard,  53  per  cent 
would  have  been  condemned  by  Houston's  standard, 
although  containing  only  resistant  forms  which  Clemesha 


VAEIETIES  OF  COLON  BACILLI  197 

believes  to  be  unimportant,  and  16  per  cent  would  be 
condemned  by  Clemesha  as  containing  true  B.  communis. 

Major  Clemesha  does  not  claim  that  these  results 
necessarily  indicate  any  change  of  procedure  in  dealing 
with  the  waters  of  temperate  climates.  Indeed,  the 
experience  of  English  and  American  bacteriologists 
offers  pretty  conclusive  evidence  that  waters  so  stored 
as  to  be  safe  do  not  contain  large  numbers  of  lactose- 
fermenting  organisms  of  any  type.  In  other  tropical 
countries  and  perhaps  in  warm  summer  weather,  the 
Indian  conditions  may  possibly  be  duplicated  (as  we 
know  they  are  in  the  case  of  the  forms  fermenting 
dextrose  but  not  lactose) ;  and  the  experiments  reported 
in  this  book  deserve  the  careful  consideration  of  water 
bacteriologists  and  sanitarians. 

The  results  obtained  by  Houston  (1911)  in  London 
unfortunately  do  not  correspond  at  all  with  these 
Indian  data.  Houston  studied  in  detail  the  reactions 
of  about  800  strains  of  dextrose-fermenting  bacteria 
from  raw  river-water,  stored  water,  and  stored  and 
filtered  water.  Comparison  of  the  relative  prevalence 
of  types  from  these  three  sources  ought  to  furnish  some 
confirmation  of  Major  Clemesha's  conclusions,  even 
although  the  extreme  conditions  of  warmth  and  sun- 
light are  lacking.  We  find,  however,  on  careful  study 
of  the  figures  that  they  do  not.  The  Houston  types 
corresponding  to  B.  communis,  B.  Schafferi  and  B. 
neapolitanus  (sensitive  forms)  are  on  the  whole  but 
little  more  prevalent  in  the  raw  than  in  the  stored 
and  filtered  waters.  On  the  other  hand  the  types 
corresponding  to  B.  Griinthal,  B.  vesiculosus,  B.  cos- 


198       ELEMENTS  OF  WATER  BACTERIOLOGY 


coroba  and  B.  cloacae  (Clemesha's  resistant  types) 
are  less  abundant  in  the  filtered  and  stored  than  in  the 
raw  water.  Houston's  lactose-fermenting  forms  clas- 
sified in  MacConkey's  four  great  groups  show  the  rela- 
tions indicated  in  the  table  below,  which  are  almost 
the  reverse  of  what  should  be  expected  if  the  dulcite- 
fermenting  forms  (Groups  II  and  III)  were  indicative 
of  recent  pollution. 

DISTRIBUTION    OF    MACCONKEY'S    GROUPS    IN    RAW, 
STORED,    AND    FILTERED    WATER    AT   LONDON 


Pei 

centage 

in 

Group. 

Reactions. 

Type. 

Raw 

Stored 

Filtered 

Water. 

Water. 

Water. 

I 

Saccharose  —  Dulcite  — 

B.  acidi-lactici  .  .  . 

34 

39 

37 

II 

Saccharose—  Dulcite  + 

B.  coli  

23 

25 

38 

III 

Saccharose+Dulcite+ 

B.  neapolitanus.  . 

15 

26 

9 

IV 

Saccharose  +  Dulcite  — 

B.  lactis-aerogenes 

28 

IO 

16 

Statistical  Classification  of  the  Colon  Group.  From 
a  biological  standpoint,  there  is  a  twofold  difficulty 
with  such  a  classification  as  that  of  MacConkey  and 
Jackson.  In  the  first  place  it  is  enormously  complex, 
or  soon  becomes  so,  as  new  investigators  add  new 
diagnostic  tests.  In  the  second  place,  it  is  entirely 
arbitrary  in  its  choice  of  the  order  in  which  particular 
tests  are  to  be  used  in  splitting  up  the  group.  Closely 
related  forms  may  be  widely  separated  if  they  chance 
to  differ  in  the  one  respect  first  chosen  for  dichotomic 
division. 

The  best  basis  for  a  classification  following  natural  bio- 
logical lines  seems  to  us  to  be  the  statistical  method  first 


VARIETIES  OF  COLON  BACILLI  199 

suggested  by  Andrewes  and  Horder  (1906)  and  Winslow 
and  Winslow  (1908)  in  the  study  of  the  cocci.  The 
essential  point  about  this  method  is  that  the  characters 
of  the  organisms  studied  are  not  considered  indepen- 
dently, but  in  relation  to  each  other.  The  individual 
reactions  are  first  studied  quantitatively  in  a  considerable 
series  of  allied  strains,  so  that  those  types  of  reaction 
which  are  manifested  by  a  large  number  of  strains 
may  be  distinguished  from  the  rarer  intermediate 
variations.  In  the  second  place,  the  correlations  be- 
tween different  characters  are  used  as  a  basis  for  group- 
ing the  types  on  the  assumption  that  a  coincidence  in 
several  characters  indicates  a  closer  relationship  than 
any  single  character  alone. 

The  statistical  method  has  been  applied  to  the  colon 
group  in  two  extensive  investigations,  neither  of  which 
has  yet  been  published  in  full.  Of  the  first  by  Howe,  a 
brief,  abstract  has  appeared  (Howe,  1912).  The  second 
by  L.  A.  Rogers,  W.  M.  Clark,  and  B.  J.  Davis  we 
have  had  the  opportunity  of  seeing  in  manuscript. 
These  two  papers  promise  at  last  to  lay  a  foundation 
for  a  sound  knowledge  of  the  relationships  of  the  colon 
group. 

Howe  (1912)  in  his  investigation  dealt  with  630 
strains  of  fresh  intestinal  colon  bacilli.  He  concluded 
from  his  exhaustive  study  that  in  bacilli  of  this  type 
isolated  directly  from  stools,  the  characters  of  motility, 
indol  formation,  ammonia  production,  nitrate  reduction, 
fermentation  of  mannite,  dulcite,  and  starch  were  not 
sufficiently  correlated  with  each  other  or  with  other 
characters  to  be  of  classificatory  value.  Dextrose, 


200       ELEMENTS  OF  WATER  BACTERIOLOGY 

lactose,  saccharose  and  raffinose  he  found  to  constitute 
a  natural  metabolic  gradient,  in  the  order  named, 
fermentation  of  any  member  of  the  series  implying 
fermentation  of  those  preceding  it.  Fifty-three  per  cent 
of  his  strains  fermented  all  four  sugars,  5  per  cent  all 
but  rafnnose,  41  per  cent  attacked  dextrose  and  lactose 
only,  and  i  per  cent  dextrose  alone. 


CHAPTER  IX 
OTHER  INTESTINAL  BACTERIA 

IT  would  be  an  obvious  advantage  if  the  evidence  of 
sewage  contamination,  furnished  by  the  presence  of 
the  colon  group,  could  be  reinforced  and  confirmed 
by  the  discovery  in  water  of  other  forms  equally  char- 
acteristic of  the  intestinal  canal.  The  attention  of  a 
few  bacteriologists  in  England  and  America  has  been 
turned  in  this  direction  during  the  past  few  years;  and 
two  groups  of  organisms,  the  sewage  streptococci 
and  the  anaerobic  spore-bearing  bacilli,  have  been 
described  as  probably  significant. 

Significance  of  the  Sewage  Streptococci.  The  term 
"  sewage  streptococci,"  as  generally  used,  covers  an 
ill-defined  group,  including  many  cocci  which  do  not 
occur  in  well-marked  chains.  Those  most  commonly 
found  grow  feebly  on  the  surface  of  ordinary  nutrient 
agar,  producing  faint  transparent,  rounded  colonies, 
but  under  semi-anaerobic  conditions  flourish  better, 
giving  a  well-marked  growth  along  the  gelatin  stab 
and  only  a  small  circumscribed  film  on  the  surface. 
They  are  favored  by  the  presence  of  the  sugars  and 
ferment  dextrose  and  lactose,  with  the  formation 
of  abundant  acid  but  no  gas.  They  are  seen  under 
the  microscope  as  cocci,  occurring  as  a  rule  in  pairs, 

201 


202       ELEMENTS  OF  WATER  BACTERIOLOGY 

short  chains,  or  irregular  groups.  They  do  not  show 
visible  growth  and  do  not  form  indol  and  nitrite 
in  the  standard  peptone  and  nitrate  solutions;  most 
of  them  do  not  liquefy  gelatin,  though  occasionally 
forms  are  found  which  possess  this  power.  Until  recently 
no  systematic  study  of  the  various  species  found  in  the 
intestine  had  been  made  and  all  cocci  giving  the  char- 
acteristic growth  on  agar  and  strongly  fermenting 
lactose  are  commonly  included  as  "  sewage  streptococci." 
Although  the  significance  of  the  streptococci  as  sewage 
organisms  is  not  established  with  the  same  defmiteness 
which  marks  our  knowledge  of  the  colon  group,  these 
forms  have  been  isolated  so  frequently  from  polluted 
sources  and  so  rarely  from  normal  ones  that  it  now  seems 
reasonable  to  regard  their  presence  as  indicative  of 
pollution.  Although  originally  reported  by  Laws  and 
Andrewes  (Laws  and  Andrewes,  1894),  their  importance 
was  not  emphasized  until  1899  and  1900,  when  Hous- 
ton (Houston,  i899b,  i9Oob)  laid  special  stress  upon 
the  fact  that  streptococci  and  staphylococci  seem  to 
be  characteristic  of  sewage  and  animal  waste,  the  former 
being,  in  his  opinion,  the  more  truly  indicative  of 
dangerous  pollution,  since  they  are  "  readily  demon- 
strable in  waters  recently  polluted  and  seemingly 
altogether  absent  from  waters  above  suspicion  of  con- 
tamination." In  six  rivers  recently  extensively  sewage- 
polluted,  he  found  streptococci  in  from  one-tenth  to 
one  ten-thousandth  of  a  c.c.  of  the  water  examined, 
although  in  some  cases  the  chemical  analysis  would  not 
have  indicated  dangerous  pollution.  On  the  other 
hand,  eight  rivers,  not  extensively  polluted,  showed 


OTHER  INTESTINAL  BACTERIA  203 

no  streptococci  in  one- tenth  of  a  c.c.,  although  the 
chemical  and  the  ordinary  bacteriological  tests  gave 
results  which  would  condemn  the  waters.  Horrocks 
(Horrocks,  1901)  found  these  organisms  in  great  abun- 
dance in  sewage  and  in  waters  which  were  known  to  be 
sewage-polluted,  but  which  contained  no  traces  of 
Bacillus  coli.  He  found  by  experiment  that  B.  coli 
gradually  disappeared  from  specimens  of  sewage  kept 
in  the  dark  at  the  temperature  of  an  outside  veranda, 
while  the  commonest  forms  which  persisted  were  varieties 
of  streptococci  and  staphylococci. 

In  America  attention  was  first  called  to  these  organisms 
by  Hunnewell  and  one  of  us  (Winslow  and  Hunnewell, 
1902*),  and  the  same  authors  later  (Winslow  and  Hunne- 
well, i902b)  recorded  the  isolation  of  streptococci  from 
25  out  of  50  samples  of  polluted  waters.  Gage  (Gage, 
1902),  from  the  Lawrence  Experiment  Station,  has 
reported  the  organisms  present  in  the  sewage  of  that 
city,  while  Prescott  (i9O2b)  has  shown  that  they  are 
abundant  in  faecal  matter  and  often  overgrow  B.  coli  in 
a  few  hours  when  inoculations  are  made  from  such 
material  into  dextrose  broth.  In  the  monograph  of 
Le  Gros  (Le  Gros,  1902)  of  the  many  streptococci 
described,  all  without  exception  were  isolated,  either 
from  the  body  or  from  sewage.  Baker  and  one  of  us 
(Prescott  and  Baker,  1904),  found  these  organisms 
present  in  each  of  50  samples  of  polluted  waters.  On 
the  other  hand,  in  the  study  of  259  samples  of  presuma- 
bly unpolluted  waters,  by  the  method  of  direct  plating, 
Nibecker  and  of  the  authors  (Winslow  and  Nibecker, 
1903)  found  streptococci  in  only  one  sample.  Clemesha 


204       ELEMENTS  OF  WATER  BACTERIOLOGY 

(191 2a)  finds  that  streptococci  in  India  are  present 
in  .0001  or  .00001  gm.  of  faeces,  but  are  rare  in  waters 
unless  very  grossly  polluted.  In  a  series  of  bottle 
experiments  in  the  laboratory  and  in  the  study  of  an 
artificially  polluted  tank  outdoors  he  showed  that  they 
disappear  very  rapidly  in  water,  within  2  or  3  days 
at  the  most.  Gordon  (1904)  showed  that  certain  strep- 
tococci are  abundant  in  normal  saliva  and  are  found 
in  air  which  has  been  exposed  to  human  pollution  but 
not  in  normal  air.  On  the  whole  there  can  be  no  doubt 
of  the  fact  that  streptococci  occur  on  the  surfaces  of 
the  human  and  animal  body  more  commonly  than 
anywhere  else  in  nature. 

Isolation  of  Sewage  Streptococci.  The  isolation  of 
these  organisms  either  from  plates  or  liquid  cultures  is 
easy.  On  the  lactose-agar  plate,  made  directly  from 
a  polluted  water,  the  colonies  of  the  streptococci  may 
generally  be  distinguished  from  those  of  other  acid- 
formers  by  their  small  size,  compact  structure,  and 
deep-red  color,  which  is  permanent,  never  changing 
to  blue  at  a  later  period  of  incubation.  Developing 
somewhat  slowly,  however,  they  may  be  overlooked 
if  present  only  in  small  numbers.  In  the  dextrose- 
broth  tube,  streptococci  will  generally  appear  in  abun- 
dance after  a  suitable  period  of  incubation.  Prescott 
and  Baker,  in  the  work  above  mentioned,  found  that 
with  mixtures  of  B.  coli  and  streptococci  in  which  the 
initial  ratios  of  the  latter  to  the  former  varied  from 
i  :  94  to  208  :  i,  the  colon  bacilli  developed  rapidly 
during  the  early  part  of  the  experiment,  reaching  a 
maximum  after  about  14  hours,  and  then  diminishing 


OTHER  INTESTINAL  BACTERIA 


205 


rapidly.  The  streptococci  first  became  apparent  after 
10  to  15  hours  and  reached  their  maximum  after  20  to 
60  hours,  according  to  the  number  originally  present. 
Applying  the  same  method  to  polluted  waters,  similar 
periodic  changes  were  observed;  nearly  pure  cultures 
of  B.  coli  were  first  obtained,  then  the  gradual  displace- 
ment of  one  form  by  the  other  took  place,  and  at  length 

RELATIVE  GROWTH  OF  B.   COLI  AND  SEWAGE  STREPTO- 
COCCI FROM  POLLUTED  WATERS  IN  DEXTROSE  BROTH 

(PRESCOTT  AND  BAKER,  1904) 


Sample  Number  

I 

2 

3 

4 

5 

6 

7 

8 

9 

10 

Red      colonies      developing  1 

from  i  c.c.  of  original  sam-  } 
pie  on  litmus  lactose  agar  J 

4 

10 

9 

5 

8 

55 

35 

460 

1250 

105 

ii 

B.  coli 

o 

20 

68 

200     185    400 

130 

332 

420 

410 

hrs. 

Strept 

0 

O            O 

o        o        o 

0            0            0 

o 

16 

B.  coli 

2OO 

76       I3O      27O      220      2IO 

140!   420 

285 

410 

Number    found, 
in  millions  per 

hrs. 

Strept 

40 

25      20      10:     45     30 

20 

210       75 

145 

cubic  centime- 
ter,       after, 

23 

B.  coli 

280 

150     385     370 

300     570 

2OO 

405 

320 

300 

growth  in  dex- 
trose broth  for 

hrs. 

Strept 

140 

85 

280     170 

300  1700 

no 

350 

370 

350 

various    peri- 
ods   

30 

B.  coli 

O 

0 

25 

no 

O      2IO 

20 

24 

105 

hrs. 

Strept 

474 

420 

480 

300 

390 

170 

400 

105 

250J 

63 

B.  coli 

0 

o 

O 

0 

0 

12 

8 

0 

0 

o 

hrs. 

Strept 

2 

0 

0 

45 

I 

2       45 

ISO 

86 

170 

First  gas  noted  after  (hrs.)..  . 

10 

10 

0 

0 

10 

8,      10 

6 

6 

8 

1 

the  streptococci  were  present  either  in  pure  culture 
or  in  great  predominance  as  shown  by  the  accompany- 
ing tables.  The  samples  of  water  were  plated  directly 
upon  litmus  lactose  agar  and  the  plates  were  incubated 
at  37°  for  24  hours,  when  the  red  colonies  were  counted. 
At  the  time  of  plating,  i  c.c.  from  each  sample  was  also 
inoculated  into  dextrose  broth  in  fermentation  tubes, 


206       ELEMENTS  OF  WATER  BACTERIOLOGY 


which  were  likewise  incubated  at  37°.  After  various 
periods,  as  indicated  by  the  tables  below,  the  tubes 
were  shaken  thoroughly  and  i  c.c.  of  the  contents 
withdrawn.  This  was  diluted  (generally  1-1,000,000,) 
with  sterile  water,  plated  on  litmus  lactose  agar  in  the 
usual  way,  and  incubated  for  24  hours.  The  colonies 
of  B.  coli  and  streptococci  were  distinguished  micro- 

RELATIVE  GROWTH  OF  B.   COLI  AND  SEWAGE  STREPTO- 
COCCI FROM  POLLUTED  WATERS  IN  DEXTROSE  BROTH 

(PRESCOTT  AND  BAKER,  1904) 


Sample  Number  

18 

M) 

20 

21 

22 

23 

24 

25 

Red  colonies  developing  from  i  c.c.  1 
of  original  sample  on  litmus  lac-  j- 
tose  agar  J 

i 

150 

25 

30 

50 

.04 
O 

170 

.  12 
O 
380 
128 

200 

•  55 
o 
330 
80 

IOO 

30 
1.6 

0 

f 

Number  found,  in  mil- 
lions per  cubic  centi- 
meter, after  growth^ 
in  dextrose  broth  for 
various  periods.  .  .  . 

7 
hrs. 

B.  coli 

.02 

— 

.OI 

Strept. 

O 

O 

i? 
hrs. 

B.  coli 

266 

TOO 

88     350 

Sio 

160 

220 

300 

Strept. 

150 

•    o!     40     140    240 

27 
hrs. 

B.  coli 

520 

610      72    700 

IOOO 

740 
4380 

7 
60 
35 

Strept. 

800 

860 

670 

IO80 
22 
22 

20 
31 

2500 

36 

66 
70 

7 
52 

3900 

40 
hrs. 

B.  coli 

O 

o 

IO 

Strept. 

252 

330    260 

16       38 

52 

hrs. 

B.  coli 

IO 

10 

27 

Strept. 

40 

16 

3-8 

4i 

25 

IO 

30 

scopically,  and  by  difference  in  color  and  general 
characters. 

The  successive  growth  of  these  two  intestinal  groups 
in  the  same  dextrose-broth  tube  suggests  the  following 
method  for  the  detection  of  both  B.  coli  and  sewage 
streptococci. 

Inoculate  the  desired  quantity  of  water,  preferably 


OTHER  INTESTINAL  BACTERIA  207 

i  c.c.,  into  dextrose  broth,  in  a  fermentation  tube, 
and  incubate  at  37°.  After  a  few  hours'  incubation 
examine  the  cultures  for  gas.  Within  2  or  3  hours' 
after  gas  formation,  is  first  evident,  plate  from  the 
broth  in  litmus  lactose  agar,  incubating  for  12  to  18 
hours  at  37°.  If  at  the  end  of  this  time  no  acid-produc- 
ing colonies  are  found,  it  is  probably  safe  to  assume  that 
there  were  no  colon  bacilli  present.  On  the  other 
hand,  if  red  colonies  are  developed,  these  must  be  fur- 
ther examined  by  the  regular  diagnostic  tests  for  B. 
coli.  After  the  first  plating  from  the  dextrose  broth, 
replace  the  fermentation  tube  in  the  incubator  and  allow 
it  to  remain  for  24  to  36  hours,  then  plate  again  on  litmus 
lactose  agar.  This  plating  should  give  a  nearly  pure 
culture  of  streptococci  if  these  organisms  were  originally 
present  in  the  water. 

Streptococci  as  Indicators  of  Recent  Pollution.  The 
comparative  relation  of  the  streptococci  and  the  colon 
bacilli  to  sewage  pollution  is  still  somewhat  uncertain. 
Houston  (Houston,  1900)  held  that  the  former  microbes 
imply  "  animal  pollution  of  extremely  recent  and  there- 
fore specially  dangerous  kind,"  and  Clemesha's  experi- 
ments led  to  the  same  conclusion.  Horrocks  (Horrocks, 
1901),  on  the  other  hand,  maintains,  largely  on  the 
strength  of  certain  experiments  with  stored  sewage, 
that  the  streptococci  persist  after  colon  bacilli  have 
disappeared  and  indicate  contamination  with  old  sewage 
which  is  not  necessarily  dangerous.  These  discordant 
results  are  probably  to  be  explained  by  the  different 
media  in  which  the  viability  of  the  bacteria  was  com- 
pared. It  seems  likely  that  in  sewage  where  there  is  a 


208       ELEMENTS  OF  WATER  BACTERIOLOGY 

large  amount  of  organic  food  material  present  the 
streptococci  may  kill  out  the  colon  bacilli  as  they  do 
in  the  fermentation  tube,  and  as  we  know  they  fre- 
quently do  in  shellfish.  This  would  explain  Horrocks' 
results.  On  the  other  hand,  there  is  good  evidence 
that  the  streptococci  are  less  resistant  than  B.  coli  to 
the  unfavorable  conditions  which  exist  in  water  of 
ordinary  organic  purity.  In  waters  of  potable  char- 
acter B.  coli  is  frequently  present  without  the  strep- 
tococcus; and  a  negative  test  for  streptococci  has 
little  significance.  A  positive  test,  on  the  other  hand, 
furnishes  valuable  confirmatory  evidence  of  pollution. 
This  evidence  is  of  course  of  special  importance  when 
through  the  activity  of  the  streptococci  themselves, 
or  from  any  other  cause  the  colon  isolation  has  yielded 
an  erroneous  negative  result. 

The  English  Committee  appointed  to  consider  the 
standardization  of  methods  for  the  bacterioscopic 
examination  of  water  (1904)  by  a  majority  vote  rec- 
ommended the  enumeration  of  streptococci,  as  a  routine 
procedure  in  sanitary  water  analysis,  but  in  this 
country  the  Committee  on  Standard  Methods  of  Water 
Analysis  (1912)  has  concluded  that  "the  information 
afforded  by  the  occurrence  of  these  organisms  seems 
to  be  of  less  value  than  in  the  case  of  B.  coli  and  it  is 
believed  that  for  the  present  at  least,  the  streptococcus 
test  is  of  subordinate  importance." 

Use  of  the  Streptococci  to  Distinguish  between  Human 
and  Animal  Pollution.  There  seems  some  reason  to 
hope  that  the  streptococci  may  prove  of  assistance  in 
the  important  task  of  differentiating  human  and  animal 


OTHER  INTESTINAL  BACTERIA  209 

pollution,  a  task  in  which  all  other  tests  have  so  far 
failed.  Unlike  the  colon  bacilli,  streptococci  from  the 
intestines  of  cattle  and  men  appear  to  belong  to  dis- 
tinct types.  The  recognition  of-  this  fact  we  owe 
primarily  to  Gordon  (1905),  who  made  an  elaborate 
study  of  the  fermentative  power  of  the  streptococci 
in  a  long  series  of  carbohydrate  media.  His  work 
and  that  of  Houston  (Houston,  1904;  Houston,  1905*, 
Houston,  i905b)  have  made  it  clear  that  the  streptococci 
of  the  herbivora  differ  from  those  found  in  the 
human  body  in  their  low  fermentative  power.  In  their 
review  of  the  genus,  Andrewes  and  Horder  (1906) 
describe  the  type  characteristic  of  the  herbivora  under 
the  name,  Str.  equinus,  and  define  it  by  its  failure  to 
ferment  lactose,  raffinose,  inulin  or  mannite,  or  to 
reduce  neutral  red.  Five  other  types  are  described 
from  the  human  mouth  and  intestine;  all  of  them 
ferment  lactose,  and  most  reduce  neutral  red  and  fer- 
ment raffinose.  The  commonest  intestinal  form  clots 
milk,  reduces  neutral  red  and  ferments  saccharose, 
salicin,  coniferin  and  mannite.  The  specific  types 
of  the  genus  Streptococcus,  grade  into  each  other  by 
almost  imperceptible  degrees,  and  streptococci  ferment- 
ing lactose  and  raffinose  and  reducing  neutral  red  are 
sometimes  found  in  bovine  faeces;  but  the  studies  made 
in  this  country  by  Winslow  and  Palmer  (1910)  confirm 
the  conclusions  of  the  English  observers  that  there  are 
specific  differences  between  the  streptococci  of  the 
human,  bovine,  and  equine  intestines.  The  most  im- 
portant of  these  results  are  indicated  in  the  table 
below: 


210       ELEMENTS  OF  WATER  BACTERIOLOGY 


COMPARATIVE     FERMENTATIVE     POWER     OF     STREPTO- 
COCCI  FROM  THE  HORSE,   THE   COW,   AND   MAN 

(WINSLOW  AND  PALMER,  1910) 


Streptococci. 

Percentage  of  Positive  Results  (300  Strains). 

Lactose. 

Raffinose. 

Mannite. 

Human  .... 

62 
8 
52 

6 

4 
28 

28 
2 

6 

Equine  

Bovine  

The  rarity  of  lactose-fermenting  streptococci  in  the 
horse  makes  it  probable  that  this  group  can  be  used  for 
distinguishing  pollution  by  street  washings  from  that 
due  to  domestic  sewage;  and  the  fact  that  a  considera- 
bly larger  proportion  of  human  strains  attack  mannite 
and  a  considerably  larger  proportion  of  bovine  strains 
ferment  raffinose  should  make  it  possible  to  use  the 
ratio  between  results  in  these  two  media  to  distinguish 
between  the  wash  from  pastures  and  cultivated  land 
and  sewage.  Clemesha  (i9i2a)  in  India  has,  however, 
obtained  very  different  results.  Out  of  115  strains  of 
streptococci  from  human  faeces  92  per  cent  belonged 
to  the  "lamirasacsal"  class  of  Houston  (acid  in  lactose, 
clot  in  milk,  acid  in  raffinose,  saccharose  and  salicin), 
and  none  acidified  mannite.  Of  39  strains  from  cow 
dung  all  belonged  either  to  this  same  "  lamirasacsal " 
class  or  to  the  "  larasacsal  "  class  (differing  only  in 
failing  to  clot  milk).  Nevertheless,  in  view  of  the 
importance  of  distinguishing  between  human  and  animal 
pollution  and  the  hopelessness  of  doing  so  by  means 
of  the  colon  group  these  different  types  of  streptococci 
well  deserve  further  study. 


OTHER  INTESTINAL  BACTERIA  211 

The  Anaerobic  Spore-forming  Bacilli.  The  English 
bacteriologists  have  ascribed  much  importance  as 
indicators  of  sewage  pollution  to  another  group  of  organ- 
isms, the  anaerobic  spore-forming  bacilli,  of  which  the 
form  described  as  B.  aerogenes  capsulatus  (Welch 
and  Nuttall,  1892)  and  now  called  B.  welchii,  and  the 
form  isolated  by  Klein  (Klein,  1898;  Klein,  1899)  in 
1895  in  the  course  of  an  epidemic  of  diarrhoea  at  St. 
Bartholomew's  Hospital,  described  under  the  name  of 
B.  enteritidis  sporogenes  (now  called  B.  sporogenes) 
are  types. 

The  procedure  originally  described  by  Klein  for 
isolating  B.  sporogenes  is  as  follows:  a  portion  of  the 
sample  to  be  examined  is  added  to  a  tube  of  sterile 
milk,  which  is  then  heated  to  80°  C.  for  10  minutes 
to  destroy  vegetative  cells.  The  milk  is  next  cooled 
and  incubated  under  anaerobic  conditions,  which  may 
be  accomplished  most  conveniently  by  Wright's  method. 
A  tight  plug  of  cotton  is  forced  a  quarter  way  down  the 
test-tube,  the  space  above  is  loosely  filled  with  pyrogallic 
acid,  a  few  drops  of  a  strong  solution  of  caustic  potash 
are  added,  and  the  tube  is  tightly  closed  with  a  rubber 
stopper.  After  18  to  36  hours  at  37°  the  appearance 
of  the  tube  will  be  characteristic  if  the  B.  sporogenes 
is  present.  "  The  cream  is  torn  or  altogether  dissociated 
by  the  development  of  gas,  so  that  the  surface  of  the 
medium  is  covered  with  stringy,  pinkish-white  masses 
of  coagulated  casein,  enclosing  a  number  of  gas-bubbles. 
The  main  portion  of  the  tube  formerly  occupied  by  the 
milk  now  contains  a  colorless,  thin,  watery  whey,  with  a 
few  casein  lumps  adhering  here  and  there  to  the  sides 


212       ELEMENTS  OF  WATER  BACTERIOLOGY 

of  the  tube.  When  the  tube  is  opened,  the  whey  has  a 
smell  of  butyric  acid  and  is  acid  in  reaction.  Under 
the  microscope  the  whey  is  found  to  contain  numerous 
rods,  some  motile,  others  motionless." 

Since  this  organism  is  not  present  in  very  large  num- 
bers, even  in  sewage,  the  test  of  a  water-supply  must 
be  made  with  large  samples,  and  the  concentration 
of  at  least  2000  c.c.  of  water  by  nitration  through  a 
Pasteur  filter  is  recommended  by  Horrocks  as  a  necessary 
prelude  (Horrocks,  1901).  The  Committee  on  Standard 
Methods  of  Water  Analysis  (1912)  recommends  the 
following  enrichment  procedure  for  the  isolation  of  B. 
sporogenes  which  avoids  physical  concentration.  Vari- 
ous dilutions  of  the  water  to  be  tested  are  incubated 
in  fermentation  tubes  containing  liver  broth  for  24  hours 
at  37°.  If  B.  sporogenes  is  present  gas  will  be  evolved 
and  a  characteristic  "  vile  odor "  will  be  produced. 
If  this  reaction  is  obtained  the  contents  of  each  posi- 
tive tube  is  transferred  to  an  Erlenmeyer  flask  or  large 
test-tube  and  heated  at  80°  C.  for  10  minutes  to  destroy 
vegetative  cells.  One  c.c.  of  broth  containing  sediment 
is  withdrawn  from  the  bottom  of  each  flask  and  enriched 
once  more  in  a  fresh  liver  broth  tube.  B.  sporogenes 
will  now  usually  be  present  in  pure  culture  showing 
large  sluggishly  motile  bacilli  containing  spores.  A 
gelatin  stab  culture  made  from  these  24-hour  liver  broth 
tubes  will  show  after  48  hours  incubation  at  20°  a  dis- 
tinct liquefying  anaerobic  growth  beginning  about  2 
cm.  below  the  surface  with  gas  bubbles  at  the  top  of  the 
liquefied  area.  In  order  to  obtain  absolutely  pure 
cultures  it  is  necessary  to  fish  from  liver  broth  tubes 


OTHER  INTESTINAL  BACTERIA  213 

only  3-5  hours  old  as  only  young  vegetative  cells  will 
grow  on  plates.  Transplants  from  the  closed  arm  of 
such  tubes  will  grow  on  dextrose  liver  agar  plates  incu- 
bated under  anaerobic  conditions. 

The  organisms  of  the  B.  sporogenes  group  are  large 
stout  bacilli  often  occurring  in  chains.  They  liquefy 
gelatin  vigorously  and  on  agar  produce  fine  discrete 
gray  colonies.  They  vigorously  ferment  dextrose,  lac- 
tose and  saccharose,  producing  acid  and  gas,  and  in  sugar 
agar  each  colony  will  be  marked  by  one  or  more  gas 
bubbles  surrounded  by  a  delicate  whitish  fringe.  The 
organism  is  strongly  pathogenic  for  guinea  pigs,  by 
which  character  it  is  distinguished  from  the  B.  butyricus 
of  Botkin.  B.  welchii  differs  from  B.  sporogenes  chiefly 
in  lacking  motility  and  in  forming  spores  with  less  read- 
iness (Klotz  and  Holman,  1911). 

The  researches  of  Klein  and  Houston  (Klein  and 
Houston,  1898,  1899)  have  shown  that  the  B.  sporogenes 
occurs  in  English  sewage  in  numbers  varying  from  30  to 
2  200  per  c.c.  and  that  it  is  often  absent  in  considerable 
volumes  of  pure  water.  In  Boston  sewage  it  may 
usually  be  isolated  from  .01  or  .001  of  a  c.c.  (Winslow 
and  Belcher,  1904).  Since  the  spores  of  an  anaerobic 
bacillus  may  persist  for  an  indefinite  period  in  polluted 
waters,  their  presence  need  not  necessarily  indicate 
recent  or  dangerous  pollution. 

Vincent  (1907)  and  other  French  observers  consider  the 
determination  of  the  total  number  of  anaerobic  bacteria 
as  significant,  since  the  decomposition  of  organic  matter 
is  accompanied  by  anaerobic  growth.  It  is  not  claimed, 
however,  that  bacteria  of  this  type  are  characteristic  of 


214       ELEMENTS  OF  WATER  BACTERIOLOGY 

animal  more  than  of  vegetable  decompositions,  and  the 
total  anaerobic  count  apparently  adds  nothing  of  impor- 
tance to  the  information  gained  by  the  ordinary  gelatin 
plate  method.  The  property  of  liquefaction  was  for- 
merly believed  to  be  of  significance,  inasmuch  as  the 
liquefying  bacteria  were  regarded  as  indicative  of  pollu- 
tion. This  position  is,  however,  no  longer  tenable, 
since  many  bacteria,  typical  of  the  purest  waters,  may 
cause  liquefaction. 

As  Savage  says  in  summing  up  this  question:  "  The 
number  of  different  species  of  organisms  in  sewage 
is  very  great,  and  it  is  highly  probable  that  many  of 
them  occur  in  all  specimens  of  ordinary  sewage;  but, 
except  for  B.  coli,  streptococci,  and  B.  enteritidis  sporo- 
genes,  their  presence  has  not  been  ascertained  with 
sufficient  constancy,  nor  has  their  numerical  occurrence 
been  sufficiently  investigated  to  make  them  of  value 
as  indicators  of  sewage  pollution."  (Savage,  1906.) 


CHAPTER  X 

THE  SIGNIFICANCE  AND  APPLICABILITY  OF  THE 
BACTERIOLOGICAL   EXAMINATION 

Sanitary  Inspection  and  Sanitary  Analysis.  The  first 
attempt  of  the  expert  called  in  to  pronounce  upon  the 
character  of  a  potable  water  should  be  to  make  a 
thorough  sanitary  inspection  of  the  pond,  stream,  well 
or  spring  from  which  it  is  derived.  Study  of  the  pos- 
sible sources  of  pollution  on  a  watershed,  of  the  direc- 
tion and  velocity  of  currents  above  and  below  ground, 
of  the  character  of  soil  and  the  liability  to  contamina- 
tion by  surface-wash  are  of  supreme  importance  in 
interpreting  the  analyses  to  be  made.  In  many  cases, 
however,  the  results  of  the  sanitary  inspection  will 
be  found  to  be  by  no  means  conclusive.  If  house  or 
barnyard  drainage  or  sewage  is  actually  seen  to  enter 
a  water  used  for  drinking  purposes  it  is  obviously 
unnecessary  to  carry  out  delicate  chemical  or  bacteri- 
ological tests  to  detect  pollution.  On  the  other  hand, 
no  reconnoissance  can  show  certainly  whether  unpurified 
drainage  from  a  cesspool  does  or  does  not  reach  a 
given  well;  whether  sewage  discharged  into  a  lake 
does  or  does  not  find  its  way  to  a  neighboring  intake; 
whether  pollution  of  a  stream  has  or  has  not  been 
removed  by  a  certain  period  of  flow.  Evidence  upon 

215 


216       ELEMENTS  OF  WATER  BACTERIOLOGY 

these  points  must  be  obtained  from  a  careful  study  of  the 
characteristics  of  the  water  in  question,  and  this  study 
can  be  carried  out  along  two  lines,  chemical  and 
bacteriological. 

Sanitary  Chemical  Analysis.  A  chemical  examina- 
tion of  water  for  sanitary  purposes  is  mainly  useful 
in  throwing  light  upon  one  point — the  amount  of  decom- 
posing organic  matter  present.  It  also  gives  an  his- 
torical picture  which  may  be  of  much  value.  Humus- 
like  substances  may  be  abundant  in  surface-waters 
quite  free  from  harmful  pollution,  but  these  are  stable 
compounds.  Easily  decomposable  bodies,  on  the  other 
hand,  must  obviously  have  been  recently  introduced 
into  the  water  and  mark  a  transitional  state.  "  The 
state  of  change  is  the  state  of  danger,"  as  Dr.  T.  M. 
Drown  once  phrased  it.  Sometimes  the  organic  mat- 
ter has  been  washed  in  by  rain  from  the  surface  of  the 
ground,  sometimes  it  has  been  introduced  in  the  more 
concentrated  form  of  sewage.  In  any  case,  it  is  a  warn- 
ing of  possible  pollution,  and  the  determination  of  free 
ammonia,  nitrites,  carbonaceous  matter,  as  shown 
by  "  oxygen  consumed,"  and  dissolved  oxygen  yield 
important  evidence  as  to  the  sanitary  quality  of  a  water. 

Furthermore,  nitrates,  the  final  products  of  the  oxida- 
tion of  organic  matter,  and  the  chlorine  introduced  as 
common  salt  into  all  water  which  has  been  in  contact 
with  the  wastes  of  human  life,  furnish  additional  infor- 
mation as  to  the  antecendents  of  a  sample.  The  results 
of  the  chlorine  determination  are  indeed  perhaps  more 
clear  than  those  of  any  other  part  of  the  analysis,  for 
chlorine  and  sewage  pollution  vary  together,  due  allow- 


BACTERIOLOGICAL  EXAMINATION  217 

ance  being  made  for  the  proximity  of  the  sea  and  other 
geological  and  meteorological  factors.  Unfortunately, 
it  is  only  past  history  and  not  present  conditions  which 
these  latter  tests  reveal,  for  in  a  ground-water  completely 
purified  from  a  sanitary  standpoint  such  soluble  con- 
stituents remain,  of  course,  unchanged.  Thus,  in  the 
last  resort,  it  is  upon  the  presence  and  amount  of  decom- 
posing organic  matter  in  the  water  that  the  opinion  of 
the  chemist  must  be  based. 

Information  Furnished  by  Bacteriological  Examina- 
tions. The  decomposition  of  organic  matter  may  be 
measured  either  by  the  material  decomposed  or  by  the 
number  of  organisms  engaged  in  carrying  out  the  proc- 
ess of  decomposition.  The  latter  method  has  the  advan- 
tage of  far  greater  delicacy,  since  the  bacteria  respond  by 
enormous  multiplication  to  very  slight  increases  in  their 
food-supply,  and  thus  it  comes  about  that  the  standard 
gelatin-plate  count  at  20°  roughly  corresponds,  in  not 
too  heavily  polluted  waters,  to  the  free  ammonia  and 
"  oxygen  consumed,"  as  revealed  by  chemical  analysis. 
If  low  numbers  of  bacteria  are  found,  the  evidence  is 
highly  reassuring,  for  it  is  seldom  that  water  could  be 
contaminated  under  natural  conditions  without  the 
direct  addition  of  foreign  bacteria  or  of  organic  matter 
which  would  condition  a  rapid  multiplication  of  those 
already  present.  The  bacteriologist  in  such  cases 
can  declare  the  innocence  of  the  water  with  justifiable 
certainty.  When  high  numbers  are  found  the  interpreta- 
tion is  less  simple,  since  they  may  exceptionally  be  due 
to  the  multiplication  of  certain  peculiar  water  forms. 
Large  counts,  however,  under  ordinary  conditions, 


218      ELEMENTS  OF  WATER  BACTERIOLOGY 

when  including  a  normal  variety  of  forms  indicate  the 
presence  of  an  excess  of  organic  matter,  derived  in  all 
probability  either  from  sewage  or  from  the  fresh  wash- 
ings of  the  surface  of  the  ground.  In  either  case  danger 
is  indicated. 

A  still  closer  measure  of  polluting  material  may  be 
obtained  from  the  numbers  of  colonies  which  develop 
on  litmus-lactose-agar  at  37°,  since  organisms  which 
thrive  at  the  body  temperature,  and  particularly  those 
which  ferment  lactose,  are  characteristic  of  the  intestinal 
tract  and  occur  but  rarely  in  normal  waters. 

Gage  (Gage,  1907)  has  shown  that  by  counts  at  20,  30, 
40,  and  50°  C.,  information  may  be  quickly  obtained 
which  is  of  great  assistance  in  judging  the  character 
of  the  water. 

"  Modern  methods  of  bacterial  examination  of  water, 
consisting  usually  of  determinations  of  the  numbers  of 
bacteria  by  means  of  plates  incubated  at  room  tempera- 
ture, and  of  tests  for  the  presence  or  absence  of  one  or 
two  specific  types,  occasionally  lead  to  an  erroneous 
interpretation  of  the  quality  of  a  water,  owing  to  the 
fact  that  they  do  not  yield  adequate  data  by  which 
abnormal  and  inaccurate  results  may  be  separated  from 
those  which  are  truly  indicative  of  purity  or  pollution. 
Furthermore,  as  several  days  must  elapse  before  the 
bacterial  tests  can  be  completed,  the  results  when 
obtained  may  have  passed  their  usefulness.  If,  however, 
we  can  so  modify  our  procedure  that  the  varied  char- 
acter of  the  bacteria  in  waters  of  different  classes  may 
be  quickly  and  accurately  recognized,  the  value  of 
bacterial  water  analysis  will  be  enormously  increased. 


BACTERIOLOGICAL  EXAMINATION  219 

Much  of  this  information  may  be  obtained  by  the  use 
of  selective  media,  selective  temperatures,  or  by  a 
proper  combination  of  the  two. 

"  By  the  use  of  litmus-lactose-agar  in  place  of  agar 
or  gelatin  we  obtain  similar  counts  of  total  bacteria, 
and  in  addition  are  able  to  separate  those  bacteria  into 
two  groups,  which  do  and  do  not  produce  acid  fermenta- 
tion of  lactose,  and  the  numbers  of  the  two  classes  of 
bacteria  so  obtained  indicate  more  completely  the 
character  of  the  water  than  would  the  numbers  of  either 
class  alone.  By  incubating  our  plates  at  temperatures 
of  30  or  40°  C.  we  are  able  to  obtain  counts  in  12  to  18 
hours,  which  counts,  while  smaller  than  those  on  plates 
incubated  for  a  longer  period  at  a  lower  temperature, 
appear  to  be  fully  as  significant.  If  we  increase  our 
number  of  determinations  by  incubating  duplicate 
plates  at  two  or  more  temperatures,  the  various  results 
and  the  ratios  between  them  furnish  a  check  upon  one 
another  in  addition  to  increasing  the  available  data 
upon  which  to  base  an  interpretation."  (Gage,  1907.) 

Finally,  the  search  for  the  Bacillus  coli  furnishes  the 
most  satisfactory  of  all  single  tests  for  f a3cal  contamina- 
tion. This  organism  is  preeminently  a  denizen  of  the 
alimentary  canal  and  may  be  isolated  with  ease  from 
waters  to  which  even  a  small  proportion  of  sewage  has 
been  added.  On  the  other  hand,  it  is  never  found 
in  abundance  in  waters  of  good  sanitary  quality,  and 
its  numbers  form  an  excellent  index  of  the  value  of 
waters  of  an  intermediate  grade.  The  streptococci 
appear  to  be  forms  of  a  similar  significance  useful  as 
yielding  a  certain  amount  of  confirmatory  evidence. 


220      ELEMENTS  OF  WATER  BACTERIOLOGY 

The  full  bacteriological  analysis  should  then  consist 
of  three  parts,  the  gelatin-plate  count,  as  an  estimate 
of  the  amount  of  organic  decomposition  in  process; 
the  total  count,  and  the  count  of  red  colonies,  on 
litmus-lactose-agar,  as  a  measure  of  the  organisms 
which  form  acids  and  thrive  at  the  body  temperature; 
and  the  study  of  a  series  of  lactose  bile  tubes  for  the 
isolation  of  colon  bacilli. 

Special  Advantage  of  the  Bacteriological  Examination. 
The  results  of  the  bacteriological  examination  have,  in 
several  respects,  a  peculiar  and  unique  significance. 
First,  this  examination  is  the  most  direct  method  of 
sanitary  water  analysis.  The  occurrence  of  nitrites 
or  free  ammonia  in  a  small  fraction  of  one  part  per 
million,  or  of  chlorine  in  several  parts  per  million,  do 
not  in  themselves  render  a  water  objectionable  or 
dangerous.  They  merely  serve  as  indicators  to  show 
that  germ-containing  and  germ-sustaining  organic  mat- 
ter is  present.  By  a  determination  of  the  chlorine 
and  study  of  the  relations  of  carbon  and  nitrogen, 
it  is  possible  to  determine  with  some  degree  of  accuracy 
whether  this  organic  matter  is  of  plant  or  animal  origin, 
and  hence  to  rate  its  objectionable  or  dangerous  char- 
acter. By  the  bacteriological  examination,  on  the 
other  hand,  we  are  able  to  determine  directly  whether 
particular  kinds  of  organisms  characteristic  of  sewage 
are,  or  are  not,  actually  present  in  the  water.  What 
we  dread  in  drinking-water  is  the  presence  of  pathogenic 
bacteria,  mainly  from  the  intestinal  tract  of  man, 
and  it  is  quite  certain  that  the  related  non-pathogenic 
bacteria  from  the  same  source  will  behave  more  nearly 


BACTERIOLOGICAL  EXAMINATION  221 

as  these  disease  germs  do  than  will  any  chemical  com- 
pounds. In  the  second  place,  the  bacteriological 
methods  are  superior  in  delicacy  to  any  others.  Klein 
and  Houston  (1898)  showed  by  experiment  with  dilu- 
tions of  sewage  that  the  colon  test  was  from  ten  to  one 
hundred  times  as  sensitive  as  the  methods  of  chemical 
analysis;  and  studies  of  the  self-purification  of  streams 
have  confirmed  their  results  on  a  practical  scale.  Thus 
in  the  Sudbury  River  it  was  found  that  while  chem- 
ical evidences  of  pollution  persisted  for  6  miles  beyond 
the  point  of  entrance,  the  bacteria  introduced  could 
be  detected  for  4  miles  further  (Woodman,  Winslow, 
and  Hansen,  1902). 

The  statement  is  sometimes  made  that  while  bac- 
teriological methods  may  be  more  delicate  for  the 
detection  of  pollution  in  surface-waters,  contamination 
in  ground-waters  may  best  be  discovered  by  the  chemical 
analysis.  That  such  is  not  the  case  has  been  well 
shown  by  Whipple  (Whipple,  1903)  who  cites  the  fol- 
lowing two  instances  in  which  the  presumptive  test 
revealed  contamination  not  shown  by  the  chemical 
analysis : 

"  A  certain  driven- well  station  was  located  in  swampy 
land  along  the  shores  of  a  stream,  and  the  tops  of  the 
wells  were  so  placed  that  they  were  occasionally  flooded 
at  times  of  high  water.  The  water  in  the  stream  was 
objectionable  from  the  sanitary  standpoint.  The  wells, 
themselves  were  more  than  100  feet  deep;  they  pene- 
trated a  clay  bed  and  yielded  what  may  be  termed  arte- 
sian water.  Tests  for  the  presence  of  Bacillus  coli  had 
invariably  given  negative  results,  as  might  be  naturally 


222       ELEMENTS  OF  WATER  BACTERIOLOGY 

expected.  Suddenly,  however,  the  tests  became  positive 
and  so  continued  for  several  days.  On  investigation  it 
was  found  that  some  of  the  wells  had  been  taken  up  to 
be  cleaned,  and  that  the  workmen  in  resinking  them  had 
used  the  water  of  the  brook  for  washing  them  down. 
This  allowed  some  of  the  brook- water  to  enter  the  system. 
It  was  also  found  that  at  the  same  time  the  water  in  the 
brook  had  been  high,  and  because  of  the  lack  of  packing 
in  certain  joints  at  the  top  of  the  wells  the  brook-water 
leaked  into  the  suction  main.  The  remedy  was  obvious 
and  was  immediately  applied,  after  which  the  tests  for 
Bacillus  coli  once  more  became  negative.  During  all 
this  time  the  chemical  analysis  of  the  water  was  not 
sufficiently  abnormal  to  attract  attention.  On  another 
occasion  a  water-supply  taken  from  a  small  pond  fed 
by  springs,  and  which  was  practically  a  large  open  well, 
began  to  give  positive  tests  for  Bacillus  coli,  and  on 
examination  it  was  found  that  a  gate  which  kept  out 
the  water  of  a  brook  which  had  been  formerly  connected 
with  the  pond  was  open  at  the  bottom,  although  it  was 
supposed  to  have  been  shut,  thus  admitting  a  contam- 
inated surface-water  to  the  supply."  Whipple  also 
calls  attention  to  the  report  on  the  Chemical  and 
Bacteriological  Examination  of  Chichester  Well-waters 
by  Houston  (Houston,  1901),  in  which  the  results  of 
chemical  and  bacteriological  examinations  of  thirty 
wells  were  compared.  It  was  found  that  the  bacteri- 
ological results  were  in  general  concordant  and  satis- 
factory. The  wells  which  were  highest  in  the  number 
of  bacteria  showed  also  the  greatest  amount  of  pollu- 
tion, as  indicated  by  the  numbers  of  B.  coli,  B.  sporo- 


BACTERIOLOGICAL  EXAMINATION 


223 


genes,  and  streptococci.  On  the  other  hand,  the  chlorine 
and  the  albuminoid  ammonia  showed  no  correspondence 
with  the  bacteriological  results. 

Vincent  (Vincent,  1905)  cites  an  interesting  case  of 
the  detection  of  progressive  pollution  of  a  ground- 
water  by  bacteriological  methods.  The  well  of  a 
military  camp  in  Algeria  showed  200  bacteria  per  c.c. 
before  the  arrival  of  a  regiment  of  troops.  Its  sub- 
sequent history  is  indicated  in  the  table  below: 

PROGRESSIVE  POLLUTION  OF  A  WELL 

(VINCENT,  1905) 


Bacteria  per  c.c. 

Bacillus  coli  per  c.c. 

Before  arrival  of  troops  

2OO 

O 

6  days  after  arrival 

77O 

o 

14  days  after  arrival  
41  days  after  arrival 

4,240 

6  060 

I 
2 

60  days  after  arrival 

14,900 

IO 

Thirdly,  negative  tests  for  Bacillus  coli  and  low  bac- 
terial counts  may  be  interpreted  as  proofs  of  the  good 
quality  of  water,  with  a  certainty  not  attainable  by  any 
other  method  of  analysis.  Many  a  surface-water  with 
reasonably  low  chlorine  and  ammonias  has  caused  epi- 
demics of  typhoid  fever;  but  it  is  impossible,  under  any 
natural  conditions  (except  perhaps  in  a  well  polluted 
with  urine)  that  a  water  could  contain  the  typhoid 
bacillus  without  giving  clear  evidence  of  pollution  in  the 
bile  tube  or  on  the  lactose-agar  plate. 

In  the  examination  of  springs,  especially  those  used 
for  domestic  supplies  at  country  houses,  the  authors  have 
found  that  the  bacteriological  examination  offers  a 


224       ELEMENTS  OF  WATER  BACTERIOLOGY 

more  delicate  and  more  certain  index  of  the  quality 
than  may  be  obtained  by  chemical  analysis.  In  a 
number  of  instances,  springs  located  in  pastures  have 
become  slightly  polluted  by  animals,  but  to  so  small 
an  extent  that  the  chemical  examination  gave  no  indi- 
cation of  trouble.  The  bacteria,  however,  increased 
greatly  in  number,  and  colon  bacilli  could  be  readily 
isolated  from  75  per  cent  of  the  i-c.c.  samples  of  a 
long  series  used  in  making  the  presumptive  test.  A 
single  case  may  suffice  as  an  illustration.  This  was  a 
spring  located  on  a  hill  in  Hopkinton,  Mass. 
The  chemical  analysis  was  as  follows: 

Color None 

Turbidity None 

Sediment None 

Odor  (hot) None 

Odor  (cold) None 

Parts  per  Million. 

Total  solids 33 .  oooo 

Loss  on  ignition 7 .  oooo 

Fixed  residue 26 .  oooo 

Hardness 1 1 .  oooo 

Chlorine 10 .  oooo 

Nitrogen  as — 

Albuminoid  ammonia o .  oooo 

Free  ammonia o .  oooo 

Nitrites o .  oooo 

Nitrates o .  oooo 

The  bacteriological  examination  showed  a  total  count 
of  375  bacteria  per  c.c.  and  a  37°  count  of  350  per  c.c. 
The  presumptive  tests  for  Bacillus  coli  showed  that 
gas-producing  organisms  were  present  in  a  majority 
of  i -c.c.  samples,  and  typical  colon  bacilli  were  isolated. 
In  this  case  the  contamination  was  brought  about  by 
cattle  gaining  access  to  the  area  immediately  surround- 


BACTERIOLOGICAL  EXAMINATION  225 

ing  the  spring;  but  the  same  conditions  might  easily 
have  led  to  infection  from  human  beings. 

Fromme  (1910)  cites  several  interesting  examples 
of  temporary  pollution  detectable  only  by  bacteri- 
ological tests.  The  most  striking  case  was  that  of  an 
artesian  well.  Its  average  bacterial  content  had  been 
38  per  c.c.  and  colon  bacilli  were  absent  from  200  c.c. 
In  May,  1908,  this  well  became  polluted  from  a  broken 
stable  drain  10  meters  away.  The  number  of  bacteria 
rose  to  4370  and  colon  bacilli  were  found  in  10  c.c.  sam- 
ples. The  source  of  pollution  was  removed,  but  the 
well  water  in  July  still  contained  7100  bacteria  and  B. 
coli  in  i  c.c.  In  September  the  number  had  fallen  to 
105  and  colon  bacilli  were  present  in  200  c.c.  In  Novem- 
ber the  bacteria  numbered  120  and  colon  bacilli  were 
absent  from  200  c.c.  At  no  time  did  chemical  tests 
give  any  indication  of  danger,  while  the  bacteriological 
data  obviously  measured  very  delicately  a  comparatively 
slight  but  real  pollution  and  its  gradual  disappearance. 

Similar  results  have  been  reported  by  Savage  and 
Bulstrode  (Savage,  1906)  in  the  examination  of  the 
water-supply  of  Bridgend. 

It  seems  to  the  writers  that  the  real  application  of 
chemistry  begins  where  that  of  bacteriology  ends.  When 
pollution  is  so  gross  that  its  existence  is  obvious  and 
only  its  amount  needs  to  be  determined,  the  bacteri- 
ological tests  will  not  serve,  on  account  of  their  exces- 
sive delicacy.  In  studying  the  heavy  pollution  of  small 
streams,  the  treatment  of  trades  wastes,  and  the 
purification  of  sewage,  the  relations  of  nitrogenous 
compounds  and  of  oxygen  compounds  are  of  prime 


226       ELEMENTS  OF  WATER  BACTERIOLOGY 

importance.  In  other  words,  when  pollution  is  to  be 
avoided,  because  the  decomposition  of  chemical  sub- 
stances causes  a  nuisance,  it  must  be  studied  by  chem- 
ical methods.  When  the  danger  is  sanitary  and  comes 
only  from  the  presence  of  bacteria,  bacteriological 
methods  furnish  the  best  index  of  pollution. 

In  the  study  of  certain  special  problems  the  para- 
mount importance  of  bacteriology  is  generally  recognized. 
The  distribution  of  sewage  in  large  bodies  of  water 
into  which  it  has  been  discharged  may  thus  best  be 
traced  on  account  of  the  ready  response  of  the  bacterial 
counts  to  slight  proportions  of  sewage,  particularly 
since  the  ease  and  rapidity  with  which  the  technique 
of  plating  can  be  carried  out  make  it  possible  to  examine 
a  large  series  of  samples  with  a  minimum  of  time  and 
trouble.  The  course  of  the  sewage  carried  out  by  the 
tide  from  the  outlet  of  the  South  Metropolitan  Dis- 
trict of  Boston  was  studied  in  this  way  by  E.  P.  Osgood 
in  1897,  and  mapped  out  by  its  high  bacterial  content 
with  greater  accuracy  than  could  be  attained  by  any 
other  method.  Some  very  remarkable  facts  have 
been  developed  by  similar  studies  as  to  the  persistence 
of  separate  streams  of  water  in  immediate  contact 
with  each  other.  Heider  showed  that  the  sewage  of 
Vienna,  after  its  discharge  into  the  Danube  River, 
flowed  along  the  right  bank  of  the  stream,  preserving 
its  own  bacterial  characteristics  and  not  mixing  per- 
fectly with  the  water  of  the  river  for  a  distance  of 
more  than  24  miles  (Heider,  1893).  Jordan  (Jordan, 
1900),  in  studying  the  self -purification  of  the  sewage 
discharged  from  the  great  Chicago  drainage  canal, 


BACTERIOLOGICAL  EXAMINATION  227 

found  by  bacteriological  analyses  that  the  Des  Plaines 
and  the  Kankakee  Rivers  could  both  be  distinguished 
flowing  along  in  the  bed  of  the  Illinois,  the  two  streams 
being  in  contact,  yet  each  maintaining  its  own  indi- 
viduality. Finally,  the  quickness  with  which  slight 
changes  in  the  character  of  a  water  are  marked  by 
fluctuations  in  bacterial  numbers  renders  the  bacteri- 
ological methods  invaluable  for  the  daily  supervision 
of  surface  supplies  or  of  the  effluents  from  municipal 
nitration  plants. 

In  the  commoner  case,  when  normal  values  obtained 
by  such  routine  analyses  are  not  at  hand,  the  problem 
of  the  interpretation  of  any  sanitary  analysis  is  a  more 
difficult  one.  The  conditions  which  surround  a  source 
of  water  supply  may  be  constantly  changing.  No  en- 
gineer can  measure  the  flow  of  a  stream  in  July  and 
deduce  the  amount  of  water  which  will  pass  in  February; 
yet  the  July  gauging  has  its  own  value  and  significance, 
so  a  single  analysis  of  any  sort  is  not  sufficient  for  all 
past  and  future  time.  If  it  gives  a  correct  picture  of 
the  hygienic  condition  of  the  water  at  the  moment 
of  examination  it  has  fulfilled  its  task,  and  this  the 
bacteriological  analysis  can  do.  The  evidence  fur- 
nished by  inspection  and  by  chemical  analysis  should 
be  sought  for  and  welcomed  whenever  it  can  be  obtained, 
yet  we  are  of  the  opinion  that,  on  account  of  their 
directness,  their  delicacy,  and  their  certainty,  the 
bacteriological  methods  should  least  of  all  be  omitted. 


CHAPTER  XI 

BACTERIOLOGY  OF  SEWAGE  AND  SEWAGE  EFFLUENTS 

Bacteriological  and  Chemical  Examination  of  Sewage. 

The  first  object  of  modern  sewage  disposal  is  the  oxida- 
tion of  putrescible  organic  matter.  Chemical,  rather 
than  bacterial,  purification  is  usually  the  prime  requisite; 
and  chemical  tests  therefore  serve  best  as  criteria  of 
the  results  obtained.  Bacteria  are  the  agents  in  the 
process  of  sewage  purification;  but  the  most  generally 
useful  measure  of  the  work  accomplished  is  the  chemical 
oxidation  attained.  "  To  employ  a  simile,  it  is  a  case 
of  the  saw  and  the  2 -foot  rule — the  saw  will  do  the 
cutting,  but  the  rule  will  measure  the  work  cut." 
(W.  J.  Didbin.) 

In  certain  cases,  however,  bacterial  as  well  as  chemical 
purity  must  be  effected,  in  view  of  special  local  require- 
ments. The  sewage  from  a  contagious  disease  hospital, 
for  example,  should  be  freed  from  infectious  material  as 
a  factor  of  safety.  Sewage  discharged  into  a  body  of 
water  adapted  for  bathing  may  well  be  so  treated  as  to 
protect  those  using  the  water.  In  the  case  of  seaboard 
cities  where  sewage  effluents  are  likely  to  contaminate 
oyster  beds  and  other  layings  of  edible  shellfish  the 
problem  assumes  great  importance.  Where  bacterially 
impure  effluents  are  discharged  into  streams  used  for 

228 


BACTERIOLOGY  OF  SEWAGE  229 

sources  of  water-supply  the  town  taking  water  may 
protect  itself  by  nitration.  It  should  so  protect  itself, 
at  any  rate,  from  the  pollution  necessarily  incident  to 
surface  waters;  and,  unless  the  bacterial  condition  of  a 
stream  or  lake  is  made  very  materially  worse  by  the 
discharge  of  sewage  effluents,  it  is  fair  that  the  respon- 
sibility of  purification  should  rest  on  the  water  works, 
rather  than  on  the  sewage  purification  plant.  Shell- 
fish, on  the  other  hand,  cannot  be  purified.  Either 
pollution  must  be  prevented,  or  the  industry  abandoned. 
Under  such  circumstances  sanitary  authorities  may 
rightly  demand,  as  they  have  demanded  at  Baltimore, 
that  bacteria,  as  well  as  putrescible  organic  matter, 
shall  be  removed  in  sewage  treatment.  Under  such 
circumstances  the  bacterial  control  of  purification 
plants  is  as  essential  as  in  the  case  of  water  filters. 
Methods  of  Bacteriological  Examination  of  Sewage  and 
Effluents.  In  England,  considerable  attention  has 
been  devoted  to  this  subject,  and  numerous  methods 
have  been  recommended  as  furnishing  valuable  criteria 
of  the  bacterial  quality  of  sewage  effluents.  Houston 
(i902b),  for  example,  suggests  various  tests  involving 
the  use  of  litmus  milk,  peptone  solution,  gelatin  tubes, 
and  neutral-red  broth,  as  well  as  the  inoculation  of 
animals.  He  considers  the  determination  of  the  num- 
bers of  B.  coli  and  B.  sporogenes  as  of  greatest  moment, 
while  the  identification  of  streptococci  is  of  value  in 
certain  cases  and  the  enumeration  of  liquefying  bacteria, 
spore-forming  aerobes,  thermophilic  bacteria,  and  hydro- 
gen sulphide  producing  bacteria  is  of  subsidiary  impor- 
tance. Rideal  (1906)  has  recently  recommended  a  some- 


230       ELEMENTS  OF  WATER  BACTERIOLOGY 

what  less  extensive  series  of  tests,  including  aerobic 
and  anaerobic  counts,  both  at  20  and  37°,  with  the 
determination  of  the  number  of  liquefiers  and  the  num- 
ber of  spore-formers.  The  results  attained  do  not 
seem  to  warrant  any  such  elaborate  procedure.  As 
far  as  the  authors  are  aware,  the  determination  of 
liquefying  bacteria,  anaerobic  bacteria  and  thermophilic 
bacteria  does  not  add  any  information  of  material 
importance  to  that  obtained  from  the  total  count. 
Some  test  for  specific  sewage  organisms  is  of  course 
desirable.  Here  again,  however,  the  determination 
of  B.  sporogenes  and  sewage  streptococci  tells  the 
observer  little  more  than  can  be  learned  from  the  routine 
use  of  the  colon  test.  In  the  United  States  the  practise 
of  sewage  bacteriologists  is  crystallizing  around  the 
total  count  and  the  estimation  of  B.  coli.  In  the  absence 
of  evidence  as  to  the  specific  value  of  other  data,  the 
routine  control  of  filter  plants  may  well  be  limited 
to  these  two  determinations. 

The  total  count  of  bacteria  should  be  made,  as  in 
the  case  of  waters,  at  20°.  Determinations  carried 
out  in  duplicate  at  37°  give  additional  information  of 
considerable  value.  The  ratio  of  the  37°  count  to  the 
20°  count  varies  with  different  sewages.  At  Boston 
the  body  temperature  count  is  70  to  80  per  cent  of 
the  total  count;  at  Lawrence  it  appears  to  be  propor- 
tionately much  lower  (Gage,  1906).  In  using  either 
medium,  it  is  well  to  add  lactose  and  litmus  and  note 
the  number  of  red  colonies,  as  a  check  on  the  enumera- 
tion of  B.  coli. 

It  should  be  borne  in  mind,  as  Lederer  and  Bach- 


BACTERIOLOGY  OF  SEWAGE          .        231 

mann  (1911)  have  recently  pointed  out,  that  the  sampling 
error  is  a  very  serious  one  with  sewage.  Duplicate 
tests  made  at  i-minute  intervals  for  a  period  of  10 
minutes  in  their  experiments  gave  extreme  values  of 
190,000  and  550,000  per  c.c. 

The  determination  of  the  number  of  colon  bacilli 
in  sewage  and  effluents  should  furnish  an  integral  part 
of  bacteriological  sewage  analysis,  since  it  is  important 
to  know  whether  the  decrease  of  intestinal  bacteria  in 
the  process  of  purification  is  proportional  to  the  reduc- 
tion of  total  bacteria.  The  State  Sewerage  Commis- 
sion of  New  Jersey  has  adopted  this  procedure  in  its 
supervision  of  the  disposal  plants  in  that  State;  and 
the  results  seem  amply  commensurate  with  the  labor 
involved.  As  in  the  case  of  polluted  waters  the  enumera- 
tion of  B.  coli  may  be  carried  out,  either  by  the  study 
of  the  red  colonies  which  appear  on  litmus-lactose-agar 
plates  inoculated  with  the  sample  directly,  or  by  the 
use  of  a  preliminary  enrichment  process.  The  com- 
plete identification  of  B.  coli  seems  unnecessarily 
tedious,  however,  where  the  organisms  are  present 
in  such  abundance.  Some  approximate  presumptive 
method  is  indicated  here,  if  anywhere;  and  the 
experience  with  polluted  water,  reviewed  in  Chapter 
VI,  points  to  the  Jackson  bile  medium  as  the  most 
promising  one.  Experience  at  the  Sewage  Experiment 
Station  of  the  Massachusetts  Institute  of  Technology 
has  shown  that  this  presumptive  test  in  general  yields 
good  results.  As  pointed  out  above,  a  48-hour  incu- 
bation period  at  37°  is  required.  All  tubes  showing 
20  per  cent  gas  at  the  end  of  this  time  may  be  con- 


232       ELEMENTS  OF  WATER  BACTERIOLOGY 

sidered    positive    tests    for    the   colon   group,  without 
serious  error. 

Numbers  of  Bacteria  in  Sewage.  The  total  number 
of  bacteria  and  the  number  of  colon  bacilli  naturally 
vary  widely  in  the  sewages  of  different  cities  and  towns. 
European  sewages,  being  more  concentrated,  show 
as  a  rule  higher  numbers  than  are  found  in  America. 
Results  compiled  from  various  sources  show  from 
1,000,000  to  5,000,000  bacteria  in  the  sewages  of  Essen, 
Berlin,  Charlottenburg,  Leeds,  Exeter,  Chorley,  and 
Oxford,  2,000,000  to  10,000,000  in  the  sewages  of  Lon- 
don, Walton,  and  W.  Derby  and  over  10,000,000  in  the 
sewages  of  Paris,  Ballater  and  Belfast  (Winslow,  1905). 
The  number  of  colon  bacilli  in  English  sewages  varies 
from  50,000  to  750,000.  In  American  sewages,  on  the 
other  hand,  bacteria  are  somewhat  less  numerous. 
At  Lawrence  the  determinations  made  from  1894  to 
1901  showed  on  the  average  2,800,000  bacteria  per  c.c. 
At  Worcester,  Eddy  reported  3,712,000  in  1901  (Eddy, 
1902);  at  Ames,  Iowa,  Walker  (1901)  found  1,248,256 
in  the  same  year.  At  Columbus,  Johnson  (1905) 
reports  an  average  of  3,600,000  bacteria  per  c.c.;  the 
individual  numbers  varied  from  320,000  to  27,000,000. 
The  number  of  colon  bacilli  varied  from  50,000  to 
1,000,000  and  averaged  500,000.  Day  samples  of 
Boston  sewage  collected  three  times  a  week,  from 
October,  1906,  to  April,  1907,  showed  an  average  of 
1,200,000  bacteria  per  c.c.  In  the  summer  months 
numbers  are  notably  higher  than  at  other  seasons 
in  many  sewages.  Thus  in  1903,  Boston  sewage  con- 
tained 2,995,000  bacteria  in  July,  4,263,600  in  August, 


BACTERIOLOGY  OF  SEWAGE  233 

11,487,500  in  September,  3,693,000  in  October,  587,100 
in  November,  and  712,000  in  December  (Winslow, 
1905).  There  is  also  a  marked  diurnal  variation 
in  the  bacterial  content  of  sewage,  since  the  flow  con- 
tains a  smaller  proportion  of  intestinal  matter  at  night 
than  at  other  times.  For  example,  a  series  of  hourly 
samples  at  the  Sewage  Experiment  Station  of  the 
Massachusetts  Institute  of  Technology  showed  the 
following  results: 

BACTERIA    IN  BOSTON    SEWAGE— AVERAGES   FOR  EACH 
FOUR-HOUR  PERIOD.     AUGUST  13-14,  1903 

(WINSLOW  AND  PHELPS,  1905) 


Period. 

Bacteria  per  c.c. 

7:30-11:30  A.M  

1  1  :3O  A.M.-3  :3o  P.M  

I,8oo,OOO 
3,2OO,OOO 

3:30-7:30  P.M. 

4  600  ooo 

7  :3o-i  i  :3O  P.M  
ii  '30  P  M  -3  '30  A.M 

3,500,000 

I  OOO  OOO 

3  :3o—  7  :3O  A.M  

400,000 

It  is  evident  that  many  published  results  of  bacterial 
examinations  of  sewage  are  in  excess  of  the  average 
values,  since  they  refer  in  most  cases  to  day  samples 
only. 

Bacterial  Content  of  Sewage  Effluents.  The  bacterial 
content  of  sewage  effluents  varies  widely  according  to 
the  process  of  purification  adopted  and  the  efficiency 
of  the  particular  plant.  The  only  process  which 
yields  a  notably  purified  effluent  from  the  bacteri- 
ological standpoint  is  that  of  filtration  through  sand. 
Processes  of  this  type  when  operated  with  care  may 
give  a  bacterial  purification  well  over  99  per  cent  as 


234       ELEMENTS  OF  WATER  BACTERIOLOGY 

shown  by  bacteriological  examinations  at  the  Brockton 
(Mass.)  filters,  reported  by  Kinnicutt,  Winslow  and 
Pratt  (1910)  as  follows: 

BACTERIA  IN  SEWAGE  AND  EFFLUENTS  AT  BROCKTON, 
AVERAGE  OF  FOUR  EXAMINATIONS,  AUTUMN  OF 
1908 


Bacteria  per  c.c. 
Gelatin  20°. 

Colon  Bacilli  per 
c.c.  Lactose  Bile. 

Sewage  

3,1  ^O.OOO 

1  50  ooo 

Effluent  A  

1,900 

4.OO 

B 

6  300 

j  r 

D  

I2Z 

o 

E  

1,400 

r 

F 

•?  OOO 

j 

Such  high  efficiencies  as  this  table  indicates  are 
often  not  realized  under  the  actual  working  condi- 
tions of  a  municipal  plant.  At  Vineland,  N.  J.,  for 
example,  the  intermittent  niters  show  a  reduction 
of  90  to  95  per  cent  in  total  bacteria  and  a  somewhat 
higher  reduction  of  B.  coli.  The  results  of  three 
examinations  made  in  1906  are  given  below. 


BACTERIA  IN  SEWAGE  AND  SAND  FILTER  EFFLUENT 
AT  VINELAND,  N.  J. 

(N.  J.  STATE  SEWERAGE  COMMISSION,  1907) 


Bacteria  per  c.c. 

B.  Coli  in 

Sewage. 

Effluent. 

Sewage. 

Effluent. 

March  2  

480,000 

20,000 

O.OOOI  C.C. 

O.OI  C.C. 

July  26  

496,000 

.  6l,000 

O.OOOI   C.C. 

O.OOI   C.C. 

July  26  

511,000 

38,000 

O.OOOOI   C.C. 

O.OOI   C.C. 

BACTERIOLOGY  OF  SEWAGE 


235 


The  newer  bacterial  processes,  contact  beds,  and 
trickling  niters  naturally  show  a  much  less  satisfactory 
bacterial  removal  than  sand  nitration  beds.  In  the 
Columbus  experiments,  Johnson  (1905)  found  from 
1,000,000  to  2,000,000  bacteria  in  the  effluents  of  con- 
tact beds  and  from  750,000  to  1,900,000  in  the  effluent 
from  trickling  niters. 

At  the  experiment  station  of  La  Madeleine,  in  Lille, 
Calmette  (1907),  reports  5,000,000  bacteria  per  c.c.  in 
the  crude  sewage,  2,900,000  in  the  second  contact 
effluent  and  800,000  in  the  effluent  from  the  trickling 
bed.  Of  20,000  B.  coli  per  c.c.  applied  to  the  filters, 
the  contact  system  delivered  4000  and  the  trickling 
bed  2000  per  c.c.  The  average  results  of  examinations 
made  three  times  a  week  at  the  Sewage  Experiment 
Station  of  the  Massachusetts  Institute  of  Technology, 
during  two  different  periods,  were  as  follows: 

BACTERIA  IN  SEWAGE,   SEPTIC   EFFLUENT  AND 
TRICKLING  EFFLUENT  AT  BOSTON 

(WINSLOW  AND  PHELPS,  1907) 


Bacteria  per  c.c. 

B.  Coli. 
Positive  Tests 
in  o.oooooi 

C.C.* 

July-Sept.,  1906. 

Oct.,  I9o6-April, 
1907. 

July-Sept., 
1906. 

No. 

Per  Cent 
Reduc- 
tion. 

No. 

Per  Cent 
Reduc- 
tion. 

Per  Cent. 

Sewage  
Septic  effluent.  .  . 
Effluent       from 

1,300,000 
1,650,000 

1,200,000 
750,000 

38 

65 
66 

Inc. 

trickling  bed  .  . 
Septic  tank  and 
trickling  bed  .  . 

750,000 
750,000 

42 
42 

200,000 
180,000 

83 

85 

35 
35 

Jackson  bile  test. 


236      ELEMENTS  OF  WATER  BACTERIOLOGY 


The  following  average  data  for  two  of  the  largest 
trickling  filter  plants  in  the  United  States  are  cited 
by  Kinnicutt,  Winslow  and  Pratt  (1910). 

BACTERIAL  CONTENT  OF  SEWAGE  AND  EFFLUENTS 
FROM  TRICKLING  FILTERS 


Place. 

Period. 

Bacteria  per  c.c. 

Screened 
Sewage. 

Septic 
Effluent. 

Filter 
Effluent. 

Reading,  Pa  
Columbus,  Ohio.  .  . 

1908-1909 
1909 

3,100,000 

2,370,000 

I,8oo,OOO 
1  ,050,000 

600.0OO 
560,000 

It  is  obvious  that  effluents  of  this  character  cannot  be 
considered  satisfactory  from  the  standpoint  of  bacterial 
purification.  As  Houston  concluded,  after  a  careful 
review  of  the  subject,  "  The  different  kinds  of  bacteria 
and  their  relative  abundance  appear  to  be  very  much 
the  same  in  the  effluents  as  in  the  crude  sewage.  Thus, 
as  regards  undesirable  bacteria,  the  effluents  frequently 
contain  nearly  as  many  B.  coli,  proteus-like  germs, 
spores  of  B.  enteritidis  sporogenes  and  streptococci, 
as  crude  sewage.  In  no  case,  seemingly,  has  the  reduc- 
tion of  these  objectionable  bacteria  been  so  marked 
as  to  be  very  material  from  the  point  of  view  of  the 
epidemiologist"  (Houston,  1902^. 

Experimental  studies  with  specific  bacteria  have 
confirmed  these  conclusions.  Houston  (igo4b)  found 
that  B.  pyocyaneus  appeared  in  the  effluent  of  a  trickling 
bed  10  minutes  after  application  to  the  top  and  con- 
tinued to  be  discharged  for  10  days.  In  septic  tanks 
and  contact  beds,  the  same  germ  persisted  for  10  days. 


BACTERIOLOGY  OF  SEWAGE  237 

Rideal  (1906)  quotes  experiments  by  Pickard  at  Exeter, 
which  show  that  typhoid  bacilli  may  persist  for  2  weeks 
in  a  septic  tank  and  that  contact  bed  treatment  only 
effects  a  90  per  cent  removal  of  these  organisms. 

Disinfection  of  Sewage  Effluents.  Where  bacterial 
purity  is  required,  some  special  process  of  disinfection 
must  be  combined  with  the  contact  bed  or  the  trickling 
filter.  For  this  purpose  treatment  with  chloride  of 
lime  or  other  chemicals  is  rapidly  gaining  ground  as  an 
important  adjunct  to  bacterial  disposal  plants;  and  in 
connection  with  this  process  bacteriological  control  is 
an  essential. 

Rideal  (1906)  first  showed  at  Guildford  that  30  parts 
of  available  chlorine  per  million  would  reduce  the 
number  of  bacteria  in  crude  sewage  from  several  mil- 
lions to  50,000,  while  50  parts  would  reduce  their 
number  to  20  per  c.c.  Colon  bacilli  were  reduced  from 
one  million  per  c.c.  to  less  than  one  per  c.c.  by  30  parts 
of  chlorine.  In  septic  effluent  25  to  44  parts  of  chlorine 
per  million  reduced  B.  coli  from  two  and  a  half  to  four 
and  a  half  million  per  c.c.  to  less  than  one  per  c.c. 
With  contact  effluents  smaller  amounts  of  chlorine 
proved  efficient,  The  primary  effluent  required  20 
parts  per  million,  the  secondary  effluent  10.6  parts 
per  million  and  the  tertiary  effluent  2.5  parts  per  mil- 
lion to  reduce  the  number  of  B.  coli  so  that  this  organism 
could  not  be  isolated  in  5  c.c. 

In  this  country  Phelps  and  Carpenter  (1906)  demon- 
strated the  practical  usefulness  of  bleaching  powder 
disinfection,  at  the  Sewage  Experiment  Station  of  the 
Massachusetts  Institute  of  Technology.  As  indicated 


238       ELEMENTS  OF  WATER  BACTERIOLOGY 


in  the  table  below  smaller  amounts  of  chlorine  than 
were  used  by  Rideal  will  give  good  results  with  more 
dilute  American  sewages. 

BACTERIA  IN  TRICKLING  FILTER  EFFLUENT  BEFORE 
AND  AFTER  TREATMENT  WITH  CHLORIDE  OF  LIME 
(5  PARTS  PER  MILLION  AVAILABLE  CHLORINE) 

(PHELPS  AND  CARPENTER,  1906) 


T-Jai_ 

Bacteria  per  c.c. 

B.  Coli,  Jackson  Bile  Test. 

Before. 

After. 

Before 

O.OOOOOI   C.C. 

After 

I.O  C.C. 

IQ06 

August  1  1  .... 

270,000 

69 

+     o 

+     o 

13.... 

630,000 

41 

O        0 

+     o 

'        14  

135,000 

406 

+  + 

+     o 

'        IS--.- 

230,000 

21 

o      o 

0        O 

16.... 

250,000 

37 

+     o 

0        0 

18  

HO,OOO 

40 

0        O 

+     o 

20.... 

90,000 

54 

+     o 

0        0 

21.... 

220,000 

22 

0        O 

0        0 

23.  . 

+     o 

0        0 

Average  

24O,OOO 

86 

33%  + 

22%  + 

Average 

removal  .... 

99.96% 

99-993% 

The  success  of  chemical  disinfection  varies  with  the 
character  of  the  sewage  or  effluent  treated,  since  the 
organic  matter  present  consumes  a  certain  amount 
of  the  disinfectant  and  renders  it  inoperative.  Dis- 
cordant results  are  therefore  reported  from  different 
sources. 

An  important  series  of  experiments  tarried  out  in 
Ohio  by  Kellerman,  Pratt,  and  Kimberly  (1907) 
showed  good  results  with  sand  filter  effluents  and 


BACTERIOLOGY  OF  SEWAGE  239 

contact  effluents.  Septic  sewage,  on  the  other  hand, 
required  large  amounts  of  chlorine  to  produce  a  rea- 
sonable bacterial  reduction.  The  table  on  page  240 
shows  the  results  obtained  at  Marion,  Ohio. 

In  Germany,  on  the  other  hand,  Schumacher  (1905), 
Kranepuhl  (1907),  and  Kurpjuweit  (1907)  found  larger 
amounts  of  chlorine  necessary,  in  the  neighborhood  of 
60  parts  per  million  parts  of  sewage.  Their  tests 
were  somewhat  severe,  however,  the  criterion  of  success 
being  the  absence  of  B.  coli  in  a  large  proportion  of 
liter  samples. 

Standards  for  Sewage  Effluents.  The  science  of  sew- 
age bacteriology  is  in  its  infancy;  and  it  is  difficult  to 
give  any  general  rules  for  the  interpretation  of  bac- 
teriological examinations  designed  to  indicate  whether 
disposal  plants  are  successful  or  not.  Houston  stated 
provisionally  that  the  20°  count  should  be  under  100,000 
and  the  37°  count  under  10,000,  while  B.  coli  should 
be  absent  from  .001  c.c.  and  B.  sporogenes  from  .1  c.c. 
(Houston,  i902b).  This  standard  seems  to  us  far  too 
lenient.  Either  organic  purity  alone  is  necessary,  as 
at  many  sewage  disposal  plants,  or  a  higher  grade  of 
purity  than  this  should  be  attained.  It  seems  wisest 
at  the  present  time  to  avoid  fixing  any  general  standards 
of  purity  for  sewage  effluents.  Each  case  should 
be  judged  intelligently  on  its  own  merits.  In  general, 
however,  where  bacterial  purification  is  indicated  at  all, 
it  seems  fair  to  demand  that  the  effluent  should  be 
of  such  a  quality  as  not  to  increase  materially  the 
bacterial  content  of  the  body  of  water  into  which  it 
is  discharged. 


240       ELEMENTS  OF  WATER  BACTERIOLOGY 


BACTERIA  IN  SEPTIC  EFFLUENT,  CONTACT  EFFLUENT, 
AND  SAND  EFFLUENT  AT  MARION ,  O.,  BEFORE  AND 
AFTER  TREATMENT  WITH  CALCIUM  HYPOCHLORITE 

(KELLERMAN,  PRATT,  AND  KIMBERLY,  1907) 


Date. 

Effluent. 

Average 
Available 
Chlorine. 
Parts  per 
Million. 

Bacteria  per  c.c. 

20°  C. 

37°  C.     Total  Count. 

Untreated. 

Treated. 

Untreated. 

Treated. 

1907 
Apr.  ii 
Apr.  12 
Apr.  15 
Apr.  28 
Apr.  29 
Apr.  30 
Mar.  21 
Mar.  22 
Mar.  26 

Septic 
Septic 
Septic 
Contact 
Contact 
Contact 
Sand 
Sand 
Sand 

4-3 

6.2 

7-6 
2.9 
5-0 
4.4 
3-8 
3-o 
i-S 

850,000 
4,400,000 
6oo,OOO 
IIO,OOO 
65,000 
500,000 
49,OOO 
56,000 
70,000 

I,IOO,OOO 
550,000 
400,000 
2,500 
1,  600 
800 

570 
140 
4,OOO 

1,200,000 
850,000 
450,000 

240,000 
260,000 
190,000 

73,000 
160,000 
9,800 
7,000 
20,000 

370 
400 

ISO 
60 
1  60 

Date. 

Effluent. 

Average 
Available 
Chlorine. 
Parts  per 
Million. 

Bacteria  per  c.c. 

37°  C.     Red  Colonies. 

B.  Coli. 

Untreated. 

Treated. 

Untreated. 

Treated. 

1907 
Apr.  ii 
Apr.  12 
Apr.  15 
Apr.  28 
Apr.  29 
Apr.  30 
Mar.  21 
Mar.  22 
Mar.  26 

Septic 
Septic 
Septic 
Contact 
Contact 
Contact 
Sand 
Sand 
Sand 

4-3 
6.2 

7.6 
2.9 

5-o 
4-4 
3-8 
3-0 
i-5 

55,000 
6o,OOO 
IOO,OOO 

7,400 
15,000 
51,000 

20,000 
I5,OOO 
2O,OOO 

I,OOO 

2,000 

2,000 

Not  in  o  .  5 
"     0.5 

"        I.O 

"         I.O 
"        I.O 

In         i  .0 

IO,OOO 

21,000 
1,300 
800 

4,000 

O 

3 

0 
O 

I 

BACTEEIOLOGY  OF  SEWAGE  241 

Bacteriology  of  the  Sewage  Filters  Themselves.  Before 
leaving  the  subject  of  sewage  bacteriology,  brief 
reference  must  be  made  to  the  importance  of  bacteri- 
ological studies  in  relation  to  the  processes  of  sewage 
purification  which  bring  about  the  removal  of  the 
organic  matter  itself.  Nothing  is  more  necessary  to  the 
development  of  the  present  art  of  sewage  disposal 
than  knowledge  of  the  micro-organisms  concerned  and 
of  the  conditions  which  favor  their  activity;  but  such 
knowledge  is  woefully  deficient.  Something  is  known 
of  the  nitrifying  organisms  long  ago  discovered  by 
Winogradsky.  More  recent  work,  like  that  of  Schultz- 
Schultzenstein  (1903),  Boullanger  and  Massol  (1903) 
and  Calmette  (1905),  has  cleared  up  many  points 
concerning  these  forms;  but  much  remains  to  be  done. 
In  regard  to  the  reducing  action  of  bacteria  in  the 
septic  tank  and  contact  bed  we  are  almost  wholly  in 
the  dark.  Septic  tanks  work  well  with  some  sewages 
and  badly  with  others;  and  the  presence  or  absence 
of  the  right  bacteria  is  probably  largely  responsible 
for  the  different  results.  In  some  cases,  as  at 
Plainfield,  N.  J.,  the  seeding  of  a  tank  with  cesspool 
contents  has  produced  a  material  improvement  in 
septic  action. 

Knowledge  of  the  kinds  of  bacteria  involved  would 
make  it  possible  to  substitute  scientific  control  for 
such  empiricism  and  might  well  lead  to  improved 
methods  of  a  more  intensive  character  than  are  yet 
available.  The  work  already  done  upon  a  laboratory 
scale  furnishes  promise  of  such  results.  The  student 


242      ELEMENTS  OF  WATER  BACTEEIOLOGY 

who  wishes  to  follow  out  this  line  of  investigation 
will  find  a  good  summary  of  what  is  already  known 
of  the  hydrolysis  and  denitrification  of  nitrogenous 
bodies  and  the  decomposition  of  cellulose  and  other 
carbohydrates  in  Rideal's  "  Sewage  and  the  Bacterial 
Purification  of  Sewage  "  (1906). 

Gage  (1905)  has  made  a  suggestive  study  of  the 
bacteria  which  carry  on  the  reducing  changes  in  sewage 
which  deserves  the  study  of  all  who  are  interested  in 
the  more  theoretical  aspects  of  sewage  treatment. 
His  method  consisted  in  plating  sewages  and  effluents, 
isolating  typical  cultures  and  determining  their  power 
to  decompose  peptone  and  nitrates  with  the  produc- 
tion of  ammonia  and  free  nitrogen.  The  rate  of  gelatin 
liquefaction,  the  amount  of  nitrate  reduced,  the  amount 
of  free  ammonia  formed,  and  the  amount  of  nitrogen 
liberated  were  quantitatively  determined  for  each  culture 
thus  isolated.  The  numerical  values  obtained,  multiplied 
by  the  number  of  bacteria,  apparently  of  the  same  type, 
observed  in  the  plates,  gave  coefficients  of  the  liquefying, 
denitrifying,  ammonifying,  and  nitrogen-liberating  power 
of  the  effluent;  and  these  coefficients  may  be  considered 
as  measures  for  a  given  sample  of  the  tendency  of  the 
bacterial  flora  to  set  up  certain  changes.  The  results 
of  further  studies  made  by  Clark  and  Gage  (1905), 
on  sewages  and  on  sand,  contact,  and  trickling  effluents, 
show  that  there  may  be  important  differences  between 
various  sewages  in  this  respect  which  must  render 
their  purification  more  or  less  easy.  They  indicate 
that  the  effluents  obtained  from  intermittent  sand 


BACTERIOLOGY  OF  SEWAGE  243 

filters  in  cold  weather  contain  larger  numbers  of  ammo- 
nifying and  denitrifying  bacteria  than  appear  at  other 
seasons,  which  may  help  to  explain  the  poorly  nitrified 
effluents  obtained  in  the  winter  season.  Along  these 
lines  research  work  in  sewage  bacteriology  promises 
to  be  fruitful  of  results. 


CHAPTER  XII 

BACTERIOLOGICAL  EXAMINATION   OF    SHELLFISH 

Shellfish  and  Disease.  The  pollution  of  areas 
devoted  to  the  growing  of  shellfish  and  the  consequent 
pollution  of  the  shellfish  themselves  is  a  matter  of 
much  sanitary  importance.  Oysters,  clams  and  mussels 
are  the  shellfish  commonly  used  as  food,  and  since 
they  are  likely  to  be  eaten  in  an  uncooked  or  partially 
cooked  condition,  it  is  important  to  be  assured  as  to 
their  character  from  the  bacteriological  standpoint. 
In  their  normal  habitats,  in  clean  sea-water,  or  in  river 
estuaries  free  from  pollution,  shellfish  are  unquestionably 
free  from  dangerous  bacteria,  although  their  feeding 
habits  make  it  probable  that  the  types  of  bacteria 
indigenous  to  the  waters  in  which  they  are  found  might 
be  present  in  considerable  numbers.  With  the  pollu- 
tion of  streams  by  unpurified  sewage  the  areas  in  \vhich 
oysters  and  clams  develop  may  easily  become  infected 
by  organisms  of  intestinal  types,  and  there  is,  therefore, 
offered  an  easy  means  for  the  typhoid  bacillus  and  other 
pathogenes  to  pass  from  the  sewage  directly  into  the 
intestinal  tract  of  the  consumer  of  the  raw  oysters  or 
clams. 

The  history  of  this  subject  is  well  summarized  by 
Newlands  and  Ham  (1910),  from  whose  excellent  report 
the  following  paragraphs  are  adapted: 

244 


EXAMINATION  OF  SHELLFISH  245 

Attention  was  first  drawn  to  the  danger  from  shell- 
fish by  the  remarkable  outbreak  of  typhoid  fever  which 
occurred  in  Middletown,  Conn.,  in  1894,  as  a  result 
of  the  serving  of  raw  oysters  at  college  fraternity 
banquets.  The  oysters  used  in  this  case  were  all 
derived  from  a  certain  portion  of  Long  Island  Sound, 
where  they  had  been  put  down,  or  planted,  in  order 
to  fatten.  Investigation  showed  that  the  stream 
entering  the  Sound  at  this  point  was  highly  polluted, 
and  furthermore,  that  at  a  nearby  house  there  were 
two  severe  cases  of  typhoid  fever  from  which  the  intes- 
tinal discharges  were  turned  into  the  drain  and  thence 
into  the  stream  without  disinfection.  The  course 
of  the  passage  of  the  bacteria  from  the  patient  suffer- 
ing with  the  disease  to  the  oyster  and  so  on  to  the 
young  men  at  the  banquets  was,  therefore,  traced 
out  in  a  most  complete  and  thorough  \vay.  This 
investigation,  which  was  conducted  by  Prof.  H.  W. 
Conn,  of  Wesleyan  University,  caused  immediate  invest- 
igations to  be  set  on  foot  in  England  and  in  this  coun- 
try. Two  years  later  there  followed  a  report  by  the 
Local  Government  Board  of  Great  Britain  dealing 
with  pollution  of  shellfish  along  the  English  coast,  and 
the  matter  has  also  received  much  attention  in  this 
country. 

A  study  of  the  literature  reveals  only  a  few  references 
to  oysters  as  carriers  of  disease  germs  previous  to 
1880.  In  that  year  Cameron,  in  a  paper  entitled 
"  Oysters  and  Typhoid  Fever,"  read  before  the  British 
Medical  Association,  suggested  that  outbreaks  of  typhoid 
fever  and  cholera  might  be  caused  by  eating  oysters. 


246       ELEMENTS  OF  WATER  BACTERIOLOGY 

In  1893  Thorne-Thorne,  in  a  report  to  the  Local  Gov- 
ernment Board,  wrote  that,  in  his  opinion,  certain 
cases  of  cholera  which  had  occurred  that  year  at  various 
inland  towns  in  England  were  due  to  eating  contam- 
inated oysters  from  beds  at  Grimsby,  where  there 
had  been  a  small  cholera  epidemic.  Following  '  the 
suggestions  embodied  in  this  report  the  English  Govern- 
ment began  a  series  of  investigations  which  have  made 
many  important  additions  to  our  present  knowledge 
of  the  subject. 

In  1902  the  famous  oyster  epidemics  at  Winchester 
and  Southampton,  England,  were  proven  beyond 
reasonable  doubt  to  have  been  caused  by  contami- 
nated oysters  taken  from  grounds  at  Emsworth.  Here 
again  we  have  to  deal  with  banquets  given  in  different 
cities  where  the  only  common  source  of  infection 
appears  to  have  been  contaminated  oysters.  Of  the 
267  guests  at  these  banquets  118  were  attacked  with 
intestinal  disorders  and  21  cases  of  typhoid  fever 
developed,  5  of  which  were  fatal. 

Although  a  great  many  sensational  attacks  have 
been  made  against  oysters  as  carriers  of  disease  germs 
which  have  been  based  on  little  or  no  evidence,  the 
above-mentioned  investigations  and  others,  among 
which  might  be  mentioned  those  of  Thresh,  Marvel, 
and  Soper,  have  brought  out  sufficient  trustworthy 
evidence  to  show  that  contaminated  oysters  must  be 
considered  as  a  real  factor  in  the  dissemination  of 
typhoid  fever  and  other  water-borne  diseases.  An  esti- 
mate of  the  extent  to  which  such  illness  is  due  to 
oysters  would  be  impossible  at  the  present  time.  The 


EXAMINATION  OF  SHELLFISH  247 

Royal  Sewage  Commission  after  an  extensive  investi- 
gation on  this  subject  came  to  the  following  conclusion : 
"  After  carefully  considering  the  whole  of  the  evidence 
on  this  point,  we  are  satisfied  that  a  considerable  num- 
ber of  cases  of  enteric  fever  and  other  illness  are 
caused  by  the  consumption  of  shellfish  which  have  been 
exposed  to  sewage  contamination;  but  in  the  present 
state  of  knowledge,  we  do  not  think  it  possible  to  make 
an  accurate  numerical  statement. 

"  Moreover  an  examination  of  the  figures  which 
have  been  placed  before  us  as  regards  those  towns  in 
which  the  subject  has  been  most  carefully  studied 
shows  that  there  may  be  occasional  errors.  Indeed 
the  witnesses  themselves  recognized  that  absolutely 
accurate  figures  were  not  obtainable. 

"  We  are  far  from  denying  that  isolated  cases  may 
have  been  due  to  contaminated  shellfish,  but  we  must 
remember  that  the  possibility  of  some  of  them  being 
due  to  other  causes  cannot  be  altogether  excluded." 

In  the  above-mentioned  cases,  where  oysters  have 
been  proven  or  reasonably  suspected  of  being  the  cause 
of  disease,  it  was  found  that  the  oysters  in  ques- 
tion had  been  floated  or  grown  in  heavily  polluted 
water  where  direct  contact  with  specific  infection 
could  be  proven  or  readily  assumed.  The  Wesleyan 
epidemic  is  a  case  in  point.  Oysters  had  undoubtedly 
been  floated  in  the  contaminated  waters  at  Fair  Haven 
for  a  number  of  years  previous  to  1894  without  any 
noticeable  effect  on  the  health  of  persons  eating  them, 
but  specific  infection  of  the  water  from  two  patients 
in  a  house  near  by  was  followed  by  a  serious  epidemic. 


248       ELEMENTS  OF  WATER  BACTERIOLOGY 

Valuable  studies  of  the  relation  between  shellfish 
and  disease  have  recently  been  published  by  Bulstrode 
(1911)  and  Wilhelmi  (1911)  and  Stiles  (1912). 

Effect  of  Cookery  upon  Polluted  Shellfish.  It  should 
be  noted  that  it  is  unfortunately  not  only  raw  shellfish 
which  are  responsible  for  the  spread  of  disease.  Most 
of  the  processes  of  cookery  to  which  these  foods  are 
subjected  are  insufficient  to  destroy  pathogenic  germs. 
Clark  (1906)  found  that  clams  and  oysters  in  stews 
and  fried  and  scalloped  in  the  usual  manner  were 
generally  free  from  colon  bacilli  and  streptococci. 
With  steamed  clams,  however,  the  bacteria  present 
could  not  be  destroyed  except  by  a  temperature  high 
enough  and  prolonged  enough  to  ruin  the  clams  for 
eating.  Rickards  (1907)  confirmed  these  results  as 
to  the  danger  from  steamed  clams,  while  he  found  fried 
clams  and  clams  in  chowder  and  scalloped  oysters  to 
be  practically  sterilized.  Oyster  stew,  however,  is 
not  exposed  to  long  continued  heat  as  is  clam  chowder, 
and  fried  oysters  are  less  thoroughly  heated  than 
fried  clams  in  the  ordinary  processes  in  use.  Oysters 
in  both  of  these  forms  and  fancy  roast  oysters  still 
contained  colon  bacilli  and  streptococci.  Buchan  (1910) 
finds  that  the  ordinary  methods  of  cooking  mussels 
do  not  remove  the  risk  of  typhoid  infection. 

Bacteriological  Examination  of  Shellfish.  Without 
further  discussing  the  general  sanitary  aspects  of  the 
subject  it  is  important  to  consider  just  how  one  may 
determine  whether  the  oysters  from  a  given  region  are 
polluted  or  not.  The  methods  which  have  been 
developed  for  this  work  are  essentially  modifications 


EXAMINATION  OF  SHELLFISH  249 

of  the  methods  used  in  water  examination,  involving 
sometimes  total  counts  of  bacteria  at  different  tem- 
peratures, but  especially  the  application  of  the  various 
tests  for  the  determination  of  the  colon  bacillus,  since 
here,  as  in  water  examination,  this  organism  may  be 
taken  as  an  index  of  pollution  and  its  occurrence  in 
considerable  numbers  must  be  looked  upon  not  merely 
with  suspicion,  but  as  a  practical  proof  that  the 
supernatant  waters  are  polluted  and  that  the  shell- 
fish themselves  may  contain  organisms  of  pathogenic 
importance,  such  as  B.  typhi,  B.  dysenteries,  B.  sporo- 
genes  and  others.  Determinations  of  the  pollution 
of  the  water  above  the  beds  are  sometimes  made 
as  bearing  indirectly  and  inferentially  on  the  possi- 
bility of  the  pollution  of  the  shellfish  contained  therein. 
Results  of  the  two  determinations  are  not  always 
in  close  agreement,  however,  owing  to  the  rapidly 
changing  local  conditions  due  to  tide,  etc.  The  gen- 
eral relations  and  the  individual  variations  between 
water  and  shellfish  determinations  are  well  illus- 
trated in  the  table  on  page  250  from  the  report 
by  Newlands  and  Ham  (1910)  on  conditions  in  New 
Haven  Harbor. 

Study  of  the  methods  of  examination  of  shellfish 
has  been  conducted  with  great  care  at  the  Lawrence 
Experiment  Station  by  Gage,  at  the  Sanitary  Research 
Laboratory  at  the  Institute  of  Technology  by  Phelps, 
at  Brown  University  by  Gorham,  and  in  New  York 
by  Pease.  Other  officials  of  the  Shellfish  Commissions 
of  different  States  have  also  carried  out  investigations 
upon  this  subject.  The  Lawrence  Experiment  Station 


250       ELEMENTS  OF  WATER  BACTERIOLOGY 


BACTERIA    IN    WATER    AND    SHELLFISH,     NEW    HAVEN 

HARBOR 


Water. 

Oysters. 

Station. 

Samples 
Taken. 

Av.  Number 
Bacteria  per  c.c. 

Average 
Number 
B.  Coli  * 
per  c.c. 

Average 
Number 
B.  Coli  * 
per  c.c. 

Number 
Oyster 
Samples 

Character, 
of  Bottom. 

37°  C. 

20°  C. 

Ferry  St. 

Bridge 

12 

2  IO 

I26O 

43 

Soft 

Tomlin- 

son  Br 

15 

9IO 

2650 

34 

«  * 

No.  i  ... 

15 

510 

1680 

Si 

" 

No.  2 

15 

375 

9IO 

73 

1  < 

No.  3.  .  . 

16 

255 

835 

9 

72 

16 

" 

Buoy  10 

15 

155 

450 

10 

'  * 

No.  4.  .  ! 

IS 

160 

1720 

9 

•• 

No.  5.  .  . 

i? 

615 

1340 

74 

308 

13 

" 

Buoys.  • 

23 

3iS 

715 

15 

*  * 

Buoy  8.  . 

IS 

205 

4IO 

8 

•  ' 

No.  6.  .  . 

16 

145 

485 

8 

37 

"o" 

Seaweed 

No.  7.  .  . 

21 

215 

74° 

29 

425 

ii 

Hard 

No.  96.  . 

II 

220 

260 

7 

64 

10 

*  ' 

No.  QA.  . 

13 

100 

185 

9 

46 

IO 

11 

No.  9.  .  . 

12 

195 

200 

17 

37 

IO 

1  * 

No.  7  A  . 

II 

I2O 

240 

IO 

255 

II 

*  ' 

No.  8.  .  . 

16 

180 

270 

7 

370 

6 

No.  10.  . 

23 

300 

615 

9 

100 

i 

Soft 

No.  ii  .. 

II 

405 

5io 

8 

IO 

4 

*  * 

Buoy  6.  . 

21 

815 

1690 

9 

.  „  .  . 

Buoy  3  .  . 

17 

175 

590 

6 

291 

Hard 

No.  12.  . 

14 

620 

1190 

4 

6 

5 

*  * 

No.  13.  . 

7 

240 

I2O 

IO 

10 

8 

" 

No.  14.  . 

12 

285 

I  100 

i  — 

7 

8 

'  ' 

Buoy  4.  . 

7 

375 

I4OO 

4 

No.  15-  . 

7 

455 

1680 

i 

45 

IS 

Hard 

No.  16.  . 

14 

280 

1025 

— 

i  — 

3 

Soft 

No.  17.  . 

14 

300 

I26O 



Hard 

No.  19.  . 

i 

800 

300 

No.  20.  . 

IO 

135 

860 

_ 

IO 

8 

Hard 

Buoy  2.  . 

II 

375 

90S 

No.  22.  . 

8 

305 

560 

— 

7-3 

3 

Hard 

Buoy  i  .  . 

6 

us 

995 

— 

4 

12 

No.  18.  . 

IO 

255 

675 

— 

No.  24.  . 

4 

710 

1340 

9 

10 

Hard 

No.  23  .  . 

6 

450 

240 

— 

'  * 

No.  25  .  . 

2 

130 

IOOO 

— 

No.  26.  . 

5 

130 

465 

— 

No.  27.  . 

IO 

630 

695 



Soft 

No.  28.  . 

4 

415 

1400 

— 

4 

3 

Mud  and  sand 

No.  29.  . 

s 

370 

1700 

— 

Soft 

No.  30.  . 

5 

185 

440 

— 

— 

IS 

Hard 

No.  31.  - 

7 

320 

130 

— 

— 

IS 

*  * 

No.  32.  . 

S 

70 

1050 

— 

15 

" 

No.  33-. 

4 

485 

405 

— 

— 

IO 

*  * 

No.  34-  - 

4 

535 

495 

— 

10 

'  ' 

No.  35-  • 

4, 

120 

270 

~ 

IS 

Sticky 

*  Jackson's  lactose  bile  presumptive  test  used. 

Minus  sign  after  figure  i  indicates  that  the  average  was  less  than  i. 


EXAMINATION  OF  SHELLFISH  251 

method  was  published  in  the  Massachusetts  State 
Board  of  Health  Report  for  1905  (Clark,  1906).  This 
method  consisted  in  the  total  counts  of  bacteria  de- 
veloping at  20°  and  37°  and  the  fermentation  reaction 
in  dextrose  broth.  Experience  indicated  that  it  was 
not  merely  necessary  to  examine  the  stomach  contents 
of  the  oysters  but  the  "  shell  water  "  as  well  was  sub- 
jected to  examination.  With  the  advent  of  lactose  bile 
as  a  better  medium  for  the  development  of  B.  coli 
without  interference  with  other  types  of  bacteria,  the 
substitution  of  this  medium  for  dextrose  broth  was 
commonly  made,  and  this  is  now  one  of  the  standard 
media  employed  for  the  determination. 

It  has  been  noted  that  the  superiority  of  lactose 
bile  to  dextrose  broth  is  greatest  in  water  examinations 
when  the  water  is  most  polluted.  In  the  study  of 
shellfish  the  danger  of  overgrowths  is  even  greater 
than  in  polluted  waters,  since  the  organic  matter  in 
the  oyster  and  its  surrounding  shell  water  furnishes  a 
culture  medium  for  many  bacteria.  Streptococci  are 
particularly  abundant.  As  pointed  out  in  Chapter 
IX,  streptococci  die  out  more  rapidly  than  colon  bacilli 
in  potable  waters,  but  where  organic  matter  is  present 
in  abundance  the  former  may  survive  the  latter. 

We  have  compiled  the  table  from  results  given 
by  Clark  (1906).  It  will  be  noted  that  in  all  cases 
except  in  that  of  the  shell  water  there  is  a  consider- 
able difference  between  the  dextrose  fermentation  tests 
and  the  colon  isolations,  indicating  an  overgrowth  by 
streptococci  and  other  forms,  of  colon  bacilli  originally 
present.  The  B.  sporogenes  is  also  very  frequently 


252      ELEMENTS  OF  WATER  BACTERIOLOGY 


responsible    for    such    anomalous    results    in   shellfish 
examinations. 

COLON    BACILLI    AND    STREPTOCOCCI    IN    DIFFERENT 
PORTIONS  OF  THIRTY  CLAMS 


Per  Cent  of  Samples  Showing 

Fermenta- 
tion in 
Dextrose 

B.  Coli. 

Strepto- 
cocci. 

B.  Coli  and 
Strepto- 

Broth. 

Shell  water  

90 

83 

47 

40 

Gills  

77 

53 

o- 

I  ;; 

Stomach  (intestine)..  .  . 

55 

35 

22 

12 

Rectum  (intestine)  

82 

45 

43 

13 

Liver  

77 

18 

15 

7 

Visceral  tissue  

18 

8 

7 

2 

It  will  be  noted  from  Clark's  table  above  that  the 
shell  liquor  is  not  only  freer  from  overgrowths  than  the 
portions  of  the  body  of  the  clam,  but  that  the  propor- 
tion of  positive  reactions  is  in  each  case  higher.  Since 
the  shell  water  is  of  course  easier  to  examine  than  the 
macerated  animal,  this  is  now  generally  adopted  as  the 
standard  material  for  examination. 

Self -purification  of  Shellfish.  In  connection  with 
the  bacteriological  examination  of  shellfish  for  colon 
bacilli  certain  investigations  have  been  carried  out 
which  are  of  great  importance  from  the  commercial 
as  well  as  from  the  sanitary  standpoint.  Phelps  (1911) 
has  shown  that  oysters  which  develop  in  waters  sub- 
ject to  sewage  pollution  may  be  purified  or  entirely 
freed  from  colon  bacilli  by  the  removal  of  the  oysters 
themselves  to  waters  of  purer  character,  when,  after 


EXAMINATION  OF  SHELLFISH  253 

sufficient  time  has  elapsed,  the  oysters  will  have  cleansed 
themselves  through  their  metabolic  processes  and 
become  entirely  safe  even  for  consumption  in  the  raw 
state.  It  is  of  considerable  importance  to  determine 
the  length  of  time  necessary  for  this  self-purification 
to  take  place.  Obviously,  from  the  commercial  stand- 
point it  is  desirable  to  make  it  as  short  as  possible, 
while  from  the  sanitary  standpoint  it  must  be  long 
enough  to  insure  a  thorough  and  satisfactory  removal 
of  all  traces  of  polluted  matter.  Oyster  beds  which 
are  free  from  pollution  or  which  are  sufficiently  good 
for  the  re-laying  for  polluted  oysters  are  difficult  to 
find  and  limited  in  areas  because  of  their  nearness  to 
sources  of  pollution.  The  investigations  in  question 
were  conducted  by  Phelps  in  the  Providence  River 
and  the  upper  part  of  Narragansett  Bay.  The  oysters 
were  removed  from  heavily  polluted  regions  and  car- 
ried to  waters  which  were  practically  free  from  pollu- 
tion, where  they  were  planted.  Examinations  were 
made  from  day  to  day  in  order  to  determine  the  length 
of  time  that  these  particular  oysters  showed  pollution 
and  it  was  found  that  within  4  days  the  organisms 
of  the  colon  type  were  practically  all  eliminated. 

It  must  be  borne  in  mind  that,  if  shellfish  are  care- 
lessly opened  and  handled,  they  may  suffer  a  considerable 
additional  pollution  in  the  process,  and  may  therefore 
be  much  worse  instead  of  better  than  when  they  were 
taken.  This  is  well  brought  out  by  the  table  on  page 
254,  taken  from  a  report  by  Stiles  (1911)  in  which 
shucked  market  oysters  show  much  worse  pollution 
than  market  oysters  in  the  shell. 


254       ELEMENTS  OF  WATER  BACTERIOLOGY 


COMPARATIVE   BACTERIOLOGICAL   CONDITION  OF   MAR- 
KET OYSTERS,   SHUCKED  AND  IN  THE  SHELL 

(STILES,  191 1). 


Number 
of 
Samples. 

Average  per  c.c.  Liquor. 

Bacteria. 

B.  Coli. 

Strepto- 
cocci. 

Plain  Agar. 

Bile 

Salt 
Agar. 

25° 

37° 

37° 

Shell  oysters  
Shucked  oysters  .  . 

36 
33 

6,000 
867,000 

I,OOO 

268,000 

2OO 

45,000 

7 
74,000 

8000 

Seasonal  Variation  of  Bacteria  in  Oysters.  It  has 
been  observed  by  Gorham  (1912)  and  others  that  the 
examination  of  oysters  from  certain  regions  made  in 
the'  summer  failed  to  agree  with  the  similar  analyses 
from  the  same  beds  made  in  the  winter.  With  the 
advent  of  cold  weather  there  seems  to  be  a  great 
improvement  in  the  sanitary  quality,  so  that  oysters 
taken  from  beds  in  close  proximity  to  the  outfalls  of 
large  sewers  show  in  the  colder  months  entire  absence 
of  any  evidence  of  contamination,  judged  solely  by  the 
bacteriological  data.  Thus  Gorham  found  in  the  sum- 
mer of  1910  that  all  oysters  on  the  beds  in  the  Prov- 
idence and  Warren  Rivers  and  the  upper  part  of  Narra- 
gansett  Bay  were  so  badly  polluted  by  sewage  as  to 
be  unfit  for  jood.  Colon  bacilli  were  found  in  the 
"  shell  water  "  of  every  oyster  in  amounts  as  small  as 
.01  of  a  cubic  centimeter  or  less.  Chemical  and 
bacteriological  examination  of  the  waters  over  these 


EXAMINATION  OF  SHELLFISH  255 

beds  showed  them  to  be  heavily  sewage  polluted.  In 
December  of  the  same  year  the  analyses  of  the  oysters 
were  strikingly  different,  although  the  condition  of 
the  water  was  apparently  unchanged.  In  the  examina- 
tion five  oysters  were  selected  in  each  case  and  the 
average  total  number  of  bacteria  per  cubic  centimeter 
was  determined  and  the  presence  of  colon  bacilli  was 
tested  by  the  bile  tube  and  subsequent  isolation  and 
identification  of  the  organisms.  The  table  on  page  256 
shows  the  numbers  of  bacteria  found,  and  the  propor- 
tion of  the  five  oyster  samples  in  which  colon  bacilli 
were  present  in  cubic  centimeter  amounts  and  also 
in  o.i  and  o.oi  of  a  cubic  centimeter. 

The  conclusions  arrived  at  by  Gorham  are  that 
during  the  cold  weather  the  oysters  assume  a  condition 
of  rest  or  hibernation,  during  which  time  ciliary  move- 
ment ceases  and  the  process  of  feeding  is  suspended. 
No  organisms  are  therefore  taken  in  from  the  outside 
water  and  those  inside  the  oyster  are  gradually  elim- 
inated, so  that  the  total  number  of  organisms  is 
reduced  very  considerably  and  the  oyster  becomes 
practically  free  from  colon  bacilli. 

Standard  Methods  for  the  Examination  of  Shellfish. 
The  examination  of  shellfish  for  pollution  is  regarded 
as  of  such  importance  by  the  American  Public  Health 
Association  (1912)  that  a  committee  was  established 
to  report  upon  methods  of  examination  and  estima- 
tion of  the  numbers  of  colon  bacilli  found.  The 
following  abstract  of  the  second  report  of  this 
committee  gives  the  recommendations  for  standard 
methods  for  bacteriological  examination  of  shellfish 


256       ELEMENTS  OF  WATER  BACTERIOLOGY 


SEASONAL  VARIATION    IN  THE  BACTERIAL  CONTENT  OF 
OYSTERS 

(GORHAM,    1912) 


_"£  u,  " 

Proportion  of 

Five 

Date. 

till 

Oysters  Showing 
B.  Coli  in 

Score. 

B.  Coli 
Present  in 
Water  in 

Tempera- 
ture of 
Water 

>  rt  •*-!  •+. 

<;  w  o  o 

I  C.C. 

O.I   C.C. 

o.oic  c. 

BED  No. 


PROVIDENCE  RIVER 


Dec.  20,  1910 

IOOO 

3 

i 

o 

4 

0   01  C.C. 

-1° 

Jan.  14,  IQII 

750 

5 

3 

i 

4i 

Jan.  2<.  . 

80 

4 

3 

o 

23 

O.OI  C.C. 

1° 

Tan  27. 

23 

c 

3 

o 

32 

Feb.  10  

*o 

130 

o 

2 

2 

o 

O  * 

4 

I  .O  C.C. 

0.1° 

Feb.  28  

140 

O 

0 

0 

o 

O.OOOI  C  C. 

1° 

Mar.  ii  

2OO 

5 

4 

o 

41 

O    OI  C.C. 

1.75° 

April  14  

275 

5 

2 

o 

23 

O.OI  C.C. 

-*•      i  O 

8-5° 

April  28  

700 

5 

5 

4 

410 

O.OOOI  C.C. 

12-5° 

May  12  

I7OO 

s 

< 

< 

=;oo 

0    0001  C.C. 

15° 

BED  No.  44.    PROVIDENCE  RIVER 

o 

Jan.  7,  191  1.. 

425 

5 

5 

i 

140 

0.25° 

Feb  10 

2<co 

4 

o 

o 

4 

0° 

Feb.  28 

0 

240 

c 

i 

o 

*r 

14 

o  q° 

March  n.  .  .  . 

**f-w 

IOO 

o 
c 

2 

o 

23 

•  J 

2° 

April  14  

2IO 

O 

2 

o 

o 

2 

8  S0 

April  28  

IOOO 

C 

C 

4 

4IO 

tj    J 

ii   7<5° 

May  1  2  ..... 

I  IOO 

O 

c 

O 

c; 

A. 

AIO 

•*••*••/  0 

14   7^° 

BED  No.  204.    WARREN  RIVER 

**T  "  /  0 

Jan.  25,  1911 

600 

c 

4 

I 

5° 

0° 

Feb.  10  

140 

o 
o 

0 

0 

0 

I  .O  C.C. 

0° 

Feb.  28  

400 

o 

o 

o 

O 

O.OI  C.C. 

0-75° 

March  4  

7^0 

3* 

3* 

0* 

* 

o  7=5° 

March  n..  .  . 

/  o 

60 

O 

i 

O 
0 

0 

I 

O.OI  C.C. 

/  0 

3° 

March  14.  ... 

3400 

o 

o 

o 

O 

O.OI  C.C. 

8-75° 

April  28  

1050 

5 

5 

4 

410 

O.OI  C.C. 

13° 

BED  No.  205.    WARREN  RIVER 

Dec.  22,  1910 

250 

3 

o 

o 

3 



-i° 

Feb.  10,  1911 

325 

o 

o 

0 

o 

I  .O  C.C. 

0° 

Feb.  28  

450 

4 

2 

0 

14 

O.OI  C.C. 

1° 

March  4  

600 

2 

2 

I 

5 

o.75° 

March  1  1  .  .  .  . 

85 

2 

I 

0 

3 

O.OI  C.C. 

2° 

April  14  

325 

I 

I 

o 

2 

O.OI  C.C, 

8.25° 

April  28  

4000 

5 

5 

5 

500 

O.OI  C.C. 

ii-5° 

*  Only  three  oysters  used. 


EXAMINATION  OF  SHELLFISH  257 

which  were  adopted  by  the  Association  at  its  meet- 
ing in  1912: 

RECOMMENDATIONS    FOR    STANDARD    METHODS    FOR    THE 
BACTERIOLOGICAL  EXAMINATION  OF  SHELLFISH 

Oysters  in  the  Shell 

Selection  of  Sample.  Twelve  (12)  oysters  of  the  average 
size  of  the  lot  under  examination,  with  deep  bowls,  short 
lips,  and  shells  tightly  closed,  shall  be  picked  out  by  hand 
and  prepared  for  transportation  to  the  laboratory. 

As  complete  a  record  of  such  data  as  is  possible  to  obtain 
shall  be  made  covering  the  following  points : 

The  exact  location  of  the  bed  from  which  the  sample 
has  been  selected. 

The  depth  of  the  water  over  the  bed  at  time  of  collection. 

The  state  of  the  tide. 

The  direction  and  velocity  of  the  wind. 

Other  weather  conditions. 

The  day  and  hour  of  the  removal  of  the  stock  from  the 
water. 

The  conditions  under  which  the  stock  has  been  kept 
since  removal  from  the  water  and  prior  to  the  taking  of 
the  sample. 

The  day  and  hour  of  the  taking  of  the  sample. 

Transportation  of  the  Sample.  The  oysters  so  selected 
shall  be  packed  in  suitable  metal  or  pasteboard  containers 
of  such  size  and  shape  that  a  number  of  them  can  be 
enclosed  in  a  shipping  case  capable  of  satisfactory  refriger- 
ation by  means  of  ice.  The  important  points  in  this  con- 
nection are: 

A.  The  prevention  of  the  mixing  of  the  oyster  liquor 
of  different  samples,  and  of  the  mixing  of  the  ice-water 
with  the  oysters. 


258      ELEMENTS  OF  WATER  BACTERIOLOGY 

B.  The  icing  of  the  samples,  if  they  are  not  to  arrive 
at  the  point  of  laboratory  examination  inside  of  36  hours, 
or  if  the  outside  temperature  is  above  50°  F. 

It  is  not  necessary  to  enclose  the  oysters  in  an  absolutely 
tight  container,  providing  the  above  conditions  are  main- 
tained. 

Condition  of  Samples.  Record  shall  be  made  of  the 
general  condition  of  the  oysters  when  received,  especially 
whether  the  shells  are  open  or  closed;  of  the  presence  of 
abnormal  odors;  and  of  the  temperature  of  the  stock. 

Technical  Procedure.  The  bacteriological  examination 
shall  be  started  as  soon  as  possible  after  the  receipt  of  the 
sample. 

The  oysters  shall  be  thoroughly  cleaned  with  a  stiff 
brush  and  clean  running  water  and  then  dried.  The  edges 
of  the  shell  shall  be  passed  through  the  flame  or  burned 
with  alcohol. 

The  opening  of  the  shell  shall  be  accomplished  by  either 
of  the  following  methods: 

A .  By  the  use  of  a  sterile  oyster  knife  in  the  usual  manner. 

B.  By   drilling   through   a  flamed  portion   of   the   shell 
near   the   hinge   with   a   sterile   drill.     The   drill   shall   be 
sterilized,  and  the  site  of  the  operation  on  the  shell  shall 
be  flamed  at  least  once  during  the  drilling  process. 

Bacterial  Counts.  Bacterial  counts  shall  be  made  of 
a  composite  sample  of  each  lot  obtained  by  mixing  the 
shell  liquor  of  five  oysters.  Agar  shall  be  used  for  the 
culture  medium  and  in  general  the  procedure  shall  be  in 
accordance  with  the  method  recommended  for  the  exam- 
ination of  water  by  the  Committee  on  Standard  Methods 
of  Water  Analysis  of  the  American  Public  Health  Association. 

The  water  used  for  dilution  purposes  shall  contain  i  per 
cent  of  sodium  chloride,  in  order  to  approximate  the  natural 
salinity  of  oyster  liquor. 

The  agar  plates  shall  be  incubated  at  20°  C.  for  three 
days  and  the  colonies  then  counted. 


EXAMINATION  OF  SHELLFISH  259 

Determination  of  Bacteria  of  the  Bacillus  Coli  Group. 
The  quantitative  determination  of  the  presence  of  B.  coli 
shall  be  in  accordance  with  the  following  procedure: 

Measured  quantities  (i.o,  o.i,  o.oi  c.c.,  etc.,  or  their 
equivalents  in  dilutions)  of  the  shell  water  of  each  of  5 
oysters  selected  from  the  dozen,  shall  be  placed  in  fer- 
mentation tubes  containing  lactose  peptone  bile,  prepared 
according  to  the  method  recommended  by  the  Committee 
on  Standard  Methods  of  Water  Analysis.  These  shall  be 
incubated  for  three  days  at  37°  C.,  and  the  presence  or 
absence  of  gas  noted  daily.  For  all  ordinary  purposes  of 
routine  work  a  development  of  from  10  to  85  per  cent  of 
gas  during  this  time  period  shall  constitute  a  positive  test 
indicating  a  presumption  of  the  presence  of  at  least  one 
bacterium  of  the  Bacillus  coli  group  in  the  quantity  of  shell 
water  tested.  But  no  final  B.  coli  rating  based  on  these 
results  shall  be  used  for  official  approval  or  condemnation 
unless  positive  confirmatory  tests  for  the  presence  of  organ- 
isms of  the  B.  coli  group  shall  have  been  obtained  from 
the  tube  of  highest  or  next  highest  dilution  from  each 
oyster,  showing  the  presence  of  gas.  These  confirmatory 
tests  shall  be  begun  immediately  upon  noting  the  formation 
of  gas,  and  carried  out  in  accordance  with  the  procedure 
recommended  by  the  Committee  on  Standard  Methods  of 
Water  Analysis. 

Statement  of  Results.  The  results  of  the  bacterial 
counts  shall  be  expressed  as  Number  of  Bacteria  per  c.c. 
The  results  of  the  tests  for  B.  coli  shall  be  expressed  either 
in  the  form  of  the  following  arbitrary  numerical  system 
to  be  known  as  "  The  American  Public  Health  Association 
Method  of  Rating  Oysters  for  B.  coli;  or  in  Estimated 
Number  of  Bacteria  of  the  B.  coli  Group  per  c.c.  of  the 
Sample." 


260       ELEMENTS  OF  WATER  BACTERIOLOGY 


The  American  Public  Health  Association  Method  of  Rating 
Oysters  for  B.  Coli.* 

The  following  values  shall  be  assigned  to  the  presence 
of  bacteria  of  the  B.  coli  group  in  each  of  the  5  oysters 
examined,  these  figures  being  the  reciprocals  of  the  greatest 
dilutions  in  which  the  test  for  B.  coli  was  positive: 

If  present  in  i.o  c.c.  but  not  in  o.i  c.c.,  a  value  of  i. 
If  present  in  o.i  c.c.  but  not  in  o.oi  c.c.,  a  value  of  10. 
If  present  in  o.oi  c.c,  but  not  in  o.ooi  c.c.,  a  value 

of  100,  etc. 

The  sum  of  these  values  for  the  5  oysters  gives  the  total 
value  for  the  sample  and  this  figure  shall  be  taken  as  the 
"  rating  for  B.  coli." 

The  results  shall  be  expressed  in  the  following  tabular 
form: 

RESULTS  OF  TESTS  FOR  B.  COLI  IN  DILUTIONS 
INDICATED 


Oysters. 

I.O  C.C. 

O.I  C.C. 

O.OI  C.C. 

Numerical 
Value. 

I 

+ 

+ 

0 

IO 

2 

+ 

+ 

0 

IO 

3 

+ 

o 

0 

I 

4 

+ 

0 

o 

I 

5 

+ 

o 

0 

I 

Total,  or  rat. 

ing  for  B.  coli 

=  23 

+  =  presence  of  bacteria  of  the  B.  coli  group  in  fermentation   tube 
test  with  lactose  bile, 
o  =  failure  to  demonstrate  presence  of  bacteria  of  the  B.  coli  group. 

Estimated  Number  of  B.  Coli  per  c.c.  If  the  standard 
B.  coli  rating  above  described  is  divided  by  5,  or,  in  general, 
if  the  rating  is  divided  by  the  number  of  oysters  tested, 
the  result  will  be  approximately  the  number  of  B.  coli 

*  Where  the  term  B.  coli  is  used,  it  refers  in  all  cases  to  bacteria  of 
the  B.  coli  group  and  not  to  the  specific  prototype. 


EXAMINATION  OF  SHELLFISH 


261 


per  c,c.  of  shell  water.  Partly  because  it  does  not  do  this 
exactly,  but  also  for  simplicity  and  the  avoidance  of  fractions, 
the  method  of  stating  results  as  an  arbitrary  rating  is 
preferred  by  the  committee.  Practical  experience  with  the 
method  also  has  appeared  to  justify  this  preference. 

Illustrations  of  the  Application  of  the  Method  of  Rating 
Oysters  for  B.  Coli.  Sometimes  results  similar  to  the 
following  are  obtained,  that  is,  one  or  more  oysters  may 
show  positive  results  in  small  quantities  of  shell  water, 
while  an  equal  number  may  show  negative  results  in  larger 
quantities.  In  this  case  the  next  lower  numerical  value 
shall  be  given  to  the  positive  results  in  the  high  dilutions, 
and  such  positive  results  shall  be  considered  as  being  trans- 
ferred to  a  lower  dilution  giving  negative  results  in  another 
oyster.  This  is  done  on  the  theory  that  inconsistent  results, 
mathematically  considered,  may  follow  naturally  from  an 
unequal  distribution  of  the  bacteria  in  the  shell  water. 
This  recession  of  the  assigned  values,  however,  shall  not 
be  carried  beyond  the  point  where  the  number  of  such 
recessions  is  greater  than  the  number  of  instances  where 
other  oysters  in  the  series  failed  to  give  positive  B.  coli 
results. 

As  examples  of  the  method  of  obtaining  the  rating  for 
B.  coli,  the  following  illustrations  are  given.  They  repre- 
sent results  that  may  be  met  with  in  practice : 


CASE  A.— RESULTS  OF  B.  COLI  TESTS  IN 
INDICATED 


DILUTIONS 


Oysters. 

I.O  C.C. 

O.I  C.C. 

0.01  C.C. 

Numerical 
Value. 

I 

2 

+ 

+ 

0 

o 

IO 
10 

3 

+ 

+ 

0 

IO 

4 

+ 

0 

o 

10  (not  i) 

5 

+ 

+ 

+ 

10  (not  100) 

50=  rating 

262       ELEMENTS  OF  WATER  BACTERIOLOGY 


CASE    B.— RESULTS    OF    B.    COLI    TESTS    IN    DILUTIONS 
INDICATED 


Oysters. 

I.O  C.C. 

O.I  C.C. 

0.01  C.C. 

Numerical 
Value. 

I 

+ 

+ 

+ 

10  (not  100) 

2 

+ 

+ 

+ 

10  (not  100) 

3 

+ 

o 

0 

i 

4 

0 

0 

0 

i  (not  o) 

5 

0 

o 

0 

i  (not  o) 

23  =  rating 

CASE    C—  RESULTS    OF    B.    COLI    TESTS    IN    DILUTIONS 
INDICATED 


Oysters. 

I.O  C.C. 

O.I  C.C. 

0.01  C.C. 

Numerical 
Value. 

I 
2 

1 

+ 

0 

o 

10 
IO 

3 

+ 

+ 

+ 

IOO 

4 

+ 

-f 

+ 

10  (not  100) 

5 

+ 

o 

o 

10  (not  i) 

1  40=  rating 

Oysters  removed  from  the  Shell  (Opened  or  Shucked  Stock). 

Except  as  hereinafter  stated,  all  the  procedures  and 
requirements  for  the  examination  of  opened  oysters,  i.e., 
shucked  stock,  shall  be  those  specified  for  the  examination 
of  oysters  in  the  shell. 

Selection  and  Preparation  of  Sample.  The  stock  in  the 
container  from  which  the  sample  is  to  be  taken  shall  be 
thoroughly  mixed,  and  one  or  more  wide-mouthed  sterile 
jars  of  a  total  capacity  of  one  quart  shall  be  each  half  filled 
with  the  sample  by  means  of  a  clean  ladle  or  other  instru- 
ment sterilized  by  flaming  alcohol.  The  jar  or  jars  shall 
be  so  sealed  as  to  exclude  all  possibility  of  contamination 
from  without. 


EXAMINATION  OF  SHELLFISH  263 

Transportation  of  Samples.  When  the  time  between 
the  collection  of  the  sample  and  its  examination  exceeds 
3  hours,  or  if  the  outside  temperature  is  above  50°  F.,  the 
sample  shall  be  thoroughly  refrigerated  by  means  of  ice 
placed  around,  but  not  in,  the  sample  jars. 

Technical  Procedure.  The  bacteriological  examination 
shall  be  begun  as  soon  as  possible  after  taking  the  sample. 
The  sample  shall  be  thoroughly  shaken  at  least  25  times 
immediately  before  opening. 

Bacterial  Counts.  The  procedure  specified  for  oysters 
in  the  shell  shall  be  followed: 

Determination  of  Bacteria  of  the  Bacillus  Coll  Group. 
The  procedure  specified  for  oysters  on  the  shell  shall  be 
followed,  but  attention  is  called  to  the  fact  that  higher 
dilutions  than  -\\-§  c.c.  are  usually  required.  Triplicate 
fermentation  tubes  shall  be  inoculated  from  each  dilution 
of  the  sample. 

Statement  of  Results.  The  results  of  the  bacteriological 
examination  of  the  opened  oysters,  or  shucked  stock,  shall 
be  expressed  in  the  same  way  as  that  specified  for  oysters 
in  the  shell,  except  that  in  the  calculation  of  B.  coli  rating 
the  values  for  the  results  of  the  positive  fermentation  tests 
after  confirmation  shall  be  recorded  for  each  of  the  inocula- 
tions of  each  dilution.  In  order  that  the  rating  from  these 
triplicate  tests  may  be  compared  with  that  obtained  from 
testing  5  oysters  in  the  shell,  the  sum  of  the  values  for 
the  triplicate  tests  shall  be  multiplied  by  f .  If,  instead,  the 
sum  is  divided  by  3,  the  result  will  give  approximately  the 
number  of  B.  coli  per  c.c. 

Clams  and  Other  Shellfish 

The  methods  for  examining  clams  and  shellfish  other 
than  oysters  shall  be  those  given  above.  Certain  modi- 
fications are  necessary  in  the  method  of  handling  the  samples 
and  the  opening  of  the  shells,  etc. 


264       ELEMENTS  OF  WATER  BACTERIOLOGY 

Clams  are  more  likely  to  lose  water  during  transportation 
than  oysters.  It  is  therefore  necessary  to  take  greater 
precautions  to  separate  different  samples  of  clams  from 
each  other  than  in  the  case  of  oysters. 

In  opening  soft  clams  it  has  been  found  that  if  two 
incisions  are  made  through  the  mantle  the  shell  water 
may  be  poured  out  without  opening  the  shell. 

Hard  clams  are  more  difficult  to  open,  but  if  the  shell 
be  struck  over  the  dorsal  muscle  with  a  small  hammer 
an  opening  will  be  formed  permitting  the  insertion  of  the 
knife  to  cut  the  muscle. 

Sometimes  clams  and  other  shellfish  contain  too  little 
liquor  to  make  all  of  the  tests  above  described.  This  is 
always  the  case  when  the  shells  are  very  small.  Under 
these  conditions  the  water  from  two  or  more  shellfish  shall 
be  taken  together  and  tested  and  considered  as  one. 

Standards  of  Interpretation.  As  in  the  case  of  water 
it  is  neither  practicable  nor  desirable  to  attempt  to  formulate 
any  hard  and  fast  standard  for  passing  or  condemning 
shellfish.  It  is  very  clear  from  the  work  carried  out 
by  the  English  Commission,  and  at  Lawrence,  Boston, 
Providence  and  New  Haven  in  this  country  that  shell- 
fish from  entirely  unpolluted  regions  are  free  from  colon 
bacilli  and  that  the  proportion  of  positive  tests  for 
these  organisms  increases  with  the  increase  in  pollution. 
Just  where  to  draw  the  line,  however,  it  is  not  easy  to 
say.  According  to  Newlands  and  Ham  (1910),  the  stand- 
ards of  permissible  pollution  adopted  by  various  English 
and  American  workers  vary  from  a  positive  test  in  i 
c.c.  to  a  positive  test  in  o.i  c.c.  of  shell  liquor.  The 
Bureau  of  Chemistry  of  the  United  States  Department 
of  Agriculture  condemns  oysters  sold  in  interstate  com- 
merce which  show  three  positive  tests  out  of  five  in  o.i  c.c. 
portions,  and  the  same  standard  has  been  adopted  by  the 
Rhode  Island  Shell  Fish  Commission. 


APPENDIX 


PREPARATION    OF  CULTURE  MEDIA 

IN  view  of  the  marked  influence  upon  bacteriological 
reactions  of  variations  in  culture  media  caused  by  differ- 
ences both  in  ingredients  and  in  technique  of  preparation, 
it  is  necessary  that  uniform  methods  be  used  in  order  to 
obtain  comparable  data.  In  specifying  the  various  ingre- 
dients used  in  culture  media  it  is  the  intention  that  they 
shall  be  uniform  in  quality,  but  it  is  not  the  intention 
that  the  recommendations  as  to  ingredients  and  technical 
manipulations  shall  stand  in  the  way  of  true  progress  as 
to  improvements.  When,  however,  improved  or  modified 
methods  are  used,  the  variations  from  the  standard  methods 
shall  be  plainly  set  forth  together  with  the  reasons  for  the 
modifications. 

INGREDIENTS 

Distilled  water  shall  be  used  in  the  preparation  of  stand- 
ard culture,  media. 

Infusions  of  fresh  lean  meat,  and  not  meat  extract, 
shall  be  used  as  the  basis  of  various  media. 

Peptone  shall  be  that  of  Witte  (dry  from  meat). 

Gelatin  shall  be  the  best  French  brand,  so  called.  It 
shall  be  as  free  as  possible  from  acids  and  other  impurities, 
and  shall  be  of  such  a  character  that  a  10  per  cent  solution 

1  From  the  Report  of  the  Committee  on  Standard  Methods  of  the 
American  Public  Health  Association. 

265 


266  APPENDIX 

prepared  in  the  usual  way  shall  not  soften  when  kept  at 
a  temperature  of  25°  C. 

Commercial  agar  in  threads  shall  be  of  as  high  a  grade 
as  can  be  obtained.  Agar  may  be  purified  by  washing. 

The  various  sugars,  such  as  dextr  se,  lactose,  and  sac- 
charose, shall  be  as  nearly  as  possible  the  chemically  pure 
compounds  designated.  Unusual  effort  to  obtain  such 
sugars  is  considered  to  be  necessary. 

Glycerine  shall  be  double  distilled. 

In  place  of  litmus,  a  i  per  cent  aqueous  solution  of 
Kahlbaum's  azolitmin  may  be  used. 

Of  the  various  other  ingredients  used,  nearly  all  of  which 
are  of  a  mineral  nature,  special  effort  shall  be  made  to 
see  that  they  are  chemically  pure  products  within  the 
full  meaning  of  this  expression. 

STERILIZATION 

Sterilization  in  the  autoclave  seems  to  be  preferable  to 
that  in  flowing  steam.  Both  in  the  lowering  of  the  melting 
point  of  gelatin  and  in  the  breaking  down  of  sugar  media 
the  time  of  sterilization  has  a  greater  effect  than  does  the 
temperature  within  the  standard  limits.  It  is,  therefore, 
suggested  that  small  containers  be  used  and  that  media 
be  sterilized  in  the  autoclave  at  120°  C.  (15  pounds  pressure) 
for  15  minutes.  A  shorter  period  than  this,  in  practice, 
sometimes  results  in  incomplete  sterilization,  and  a  longer 
time  results  in  the  inversion  of  sugars  or  the  lowering  of 
the  melting  point  of  gelatin.  Agar  media  should  be  melted 
before  placing  in  the  autoclave. 

An  important  point  in  the  sterilization  of  gelatin  and 
sugar  media  is  to  have  the  sterilizer  hot  when  the  media 
are  introduced,  so  that  heating  to  the  point  of  sterilization 
will  be  accomplished  as  quickly  as  possible.  Also  when 
sterilization  is  complete  the  media  should  be  cooled  rapidly. 
This  not  only  reduces  the  time  of  heating,  thus  preserving 


APPENDIX  267 

the  gelatin  and  sugar,  but  also  assists  in  the  actual  sterili- 
zation. It  is  also  advisable  in  the  use  of  the  autoclave 
to  keep  the  small  pet  cock  at  the  bottom  partially  open 
so  that  steam  is  escaping  during  sterilization.  This  insures 
the  removal  of  all  air.  If  practicable,  store  media  at  room 
temperature  for  two  days  to  see  that  sterilization  is  complete. 
In  intermittent  sterilization,  media  shall  be  placed  on 
each  of  three  successive  days  in  streaming  steam  for  30 
minutes  after  the  steam  fills  the  sterilizer. 

REACTION 

Phenolphthalein  shall  be  the  standard  indicator  used 
in  obtaining  the  reaction  of  all  media.  Turmeric  paper 
possesses  similar  properties,  and  its  use  is  advised  where 
phenolphthalein  is  not  available. 

Titrations  and  adjustment  of  reactions  shall  be  made 
as  follows: 

Put  5  c.c.  of  the  medium  to  be  tested  into  45  c.c.  dis- 
tilled water.  Boil  briskly  one  minute.  Add  i  c.c.  of 
phenolphthalein  solution  (5  gm.  of  commercial  salt  in  i 
liter  of  50  per  cent  alcohol).  Titrate  while  hot  (preferably 
while  boiling)  with  N/20  caustic  soda.  A  faint  but  distinct 
pink  color  marks  the  true  end-point.  This  distinct  pink 
color  may  be  more  precisely  described  as  a  combination 
of  25  per  cent  of  red  (wave  length  approximately  658) 
with  75  per  cent  of  white  as  shown  by  the  disks  of  the 
color  top  (described  under  Record  of  Tints  and  Shades 
of  Apparent  Color,  p.  10  of  Standard  Methods  Report). 
In  practice,  titration  is  continued  until  the  pink  color  of 
alkaline  phenolphthalein  matches  that  of  the  fused  disks. 

All  reactions  shall  be  expressed  with  reference  to  the 
phenolphthalein  neutral  point  and  shall  be  stated  in  per- 
centages of  normal  acid  or  alkaline  solutions  required  to 
neutralize  them.  Alkaline  media  shall  be  recorded  with 
the  minus  (  — )  sign  before  the  percentage  of  normal  acid 


268  APPENDIX 

needed  for  their  neutralization,  and  acid  media  with  the 
plus  (+)  sign  before  the  percentage  of  normal  alkaline 
solution  necessary  for  their  neutralization. 

The  standard  reaction  of  culture  media  shall  be  +1.0 
per  cent.  If  it  differs  from  i  per  cent  by  more  than  0.2 
per  cent  it  should  be  readjusted. 

Wherever  reactions  other  than  the  standard  above  given 
are  used  it  shall  be  clearly  stated  in  all  results  of  bacterial 
work,  and  the  reasons  therefor  also  stated. 

STORAGE    OF    MEDIA 

It  is  recognized  by  the  committee  that  it  is  desirable 
to  prepare  media  in  large  quantities  in  order  to  guard 
against  discrepancies  in  composition;  but,  all  things  con- 
sidered, the  complications  resulting  from  the  varying 
amounts  of  heating  incident  to  withdrawing  portions  from 
time  to  time  and  tubing  it,  are  believed  to  more  than 
offset  this  advantage.  Consequently,  when  possible,  media 
shall  be  put  at  once  into  tubes  and  placed  in  cold  storage. 

To  guard  against  changes  due  to  evaporation  all  media 
not  used  promptly  shall  be  stored  in  a  moist  atmosphere, 
preferably  in  an  ice-box,  or  else  the  flask  shall  be  sealed 
by  dipping  the  cotton  plug  in  paraffin. 

NUTRIENT    BROTH 

Nutrient  broth  shall  be  prepared  as  follows:  Infuse 
500  gm.  chopped  lean  meat  24  hours  with  1000  c.c.  dis- 
tilled water  in  refrigerator.  Restore  loss  by  evaporation. 
Strain  infusion  through  cotton  flannel.  Add  i  per  cent 
peptone.  Warm  on  water  bath,  stirring  until  the  pep- 
tone is  dissolved.  Heat  over  boiling  water  (or  steam) 
bath  30  minutes.  Restore  loss  by  evaporation.  Titrate. 
Adjust  reaction  to  -fi  per  cent  by  adding  normal  hydro- 
chloric acid  or  normal  sodium  hydrate,  as  required.  Boil 


APPENDIX  269 

2  minutes  over  free  flame,  constantly  stirring.  Restore  loss 
by  evaporation.  Filter  through  absorbent  cotton  and  cotton 
flannel,  passing  the  liquid  through  until  clear.  Titrate  and 
record  final  reaction.  Tube,  using  10  c.c.  in  each  tube. 
Sterilize. 

SUGAR  BROTHS 

Sugar  broths  shall  be  prepared  in  the  same  general  manner 
as  the  standard  nutrient  broth,  with  the  addition  of  i 
per  cent  of  dextrose,  lactose,  saccharose  or  other  sugar, 
just  before  sterilizing.  The  removal  of  muscle-sugar  by 
inoculation  with  B.  coli  is  unnecessary  if  small  amounts 
of  gas  are  disregarded. 

If,  however,  test-tubes  of  small  dimensions  are  used  and 
the  presence  of  any  gas,  however  small,  is  taken  to  indicate 
gas  formation  the  removal  of  muscle  sugar  is  necessary. 

The  reaction  of  sugar  broths  shall  be  neutral  to  phenol- 
phthalein. 

Sterilization  may  be  done  in  streaming  steam,  as  usual, 
or  in  the  autoclave  at  120°  C.  (15  pounds  pressure)  for 
15  minutes.  Sterilization  in  the  autoclave  seems  to  be 
preferable  to  that  in  flowing  steam. 

For  the  routine  work  of  testing  samples  in  water  for 
B.  coli,  especially  large  volumes  of  water  are  to  be  mixed 
with  broths  of  such  strength  that  the  resulting  mixture 
will  be  one  of  normal  strength.  Dextrose  broth  made 
with  Liebig's  Beef  Extract  is  not  equal  in  effectiveness 
to  that  made  of  fresh  beef  extract  and  should  not  be  sub- 
stituted for  the  latter. 

LACTOSE   BILE 

The  lactose  bile  medium  consists  of  sterilized  undiluted 
fresh  ox  gall  (or  a  10  per  cent  solution  of  dry  fresh  ox  gall) 
to  which  has  been  added  i  per  cent  of  peptone  and  i  per 
cent  of  lactose.  The  addition  of  peptone  is  important. 


270  APPENDIX 


GELATIN 

No  gelatin  media  should  be  employed  having  a  melting 
point  below  25°  C.  The  percentage  of  gelatin  added  may 
be  increased  to  bring  the  melting  point  up  to  the  desired 
figure.  With  most  gelatin  on  the  market  n  per  cent 
seems  to  be  preferable  to  the  standard  of  10  per  cent, 
provided  the  gelatin  is  weighed  out  without  correcting  for 
contained  moisture,  as  appears  to  have  been  the  custom. 
Ten  per  cent,  or  even  20  per  cent  of  moisture  commonly 
occurs  in  laboratory  gelatin,  and  unless  this  is  taken  into 
account  in  weighing,  the  stiffness  of  the  media  is  materially 
affected  and  the  bacterial  results  obtained  considerably 
modified.  All  gelatin  should  be  tested  for  moisture  before 
using  by  drying  a  sample  for  half  an  hour  at  105°  C.  The 
stock  should  be  kept  under  uniform  conditions  in  tight 
containers,  so  that  the  percentage  of  water  present  may 
then  be  properly  accounted  for  and  the  weight  on  a  dry 
basis  be  used  in  making  up  the  medium. 

AGAR 

For  bacterial  counts  10  gm.  of  agar  per  liter  should  be 
used.  The  smaller  amount  seems  to  be  sufficient  to  carry 
the  added  water  and  the  medium  is  less  stiff.  This  appears 
to  give  higher  and  more  consistent  counts.  Fifteen  grams 
may  be  employed  for  keeping  cultures. 

North  medium  is  especially  valuable  for  keeping  cultures, 
particularly  cocci.  This  is  composed  as  follows: 

500  c.c.  Extract  of  500  gms.  of  chopped  beef. 
500  c.c.  Distilled  Water. 
10  gm.  Agar. 
20  gm.  Gelatin. 
20  gm.  Peptone. 
5  gm.  Sodium  Chloride. 
Reaction  neutral. 

It  is  well  to  determine  the  reaction  of  the  media  after 
sterilization,  as  during  this  process  the  reaction  often  changes 


APPENDIX  271 

and  the  final  results  should  correspond  to  the  acidity 
recommended  by  the  standard  methods.  What  has  been 
said  regarding  the  necessity  of  correcting  for  moisture  in 
gelatin  applies  with  equal  force  to  agar  and  for  the  same 
reasons. 

NUTRIENT   GELATIN  AND  AGAR 

Nutrient  gelatin  and  agar  shall  be  prepared  as  follows: 

Gelatin.  Agar. 

1.  Boil  15  gm.  thread  agar  in  500  c.c. 

water  for  half  an  hour  and  make 
up  weight  to  500  gm.  or  digest  for 
15  minutes  in  the  autoclave.     Let 
this  cool  to  about  60°  C. 

2.  Infuse  500  gm.  lean  meat  24      Infuse  500  gm.  lean  meat  24  hours 
hours  with   1000  c.c.  of  dis-      with  500  c.c.  of  distilled  water  in 
tilled  water  in  refrigerator.  refrigerator. 

3.  Make  up  any  loss  by  evaporation. 

4.  Strain  infusion  through  cotton  flannel. 

5.  Weigh  filtered  infusion. 

6.  Add  i  per  cent  Witte's  pep-      Add  2  per  cent  of  Witte's   pep- 
tone  and    10   per   cent  Gold      tone. 

Label    sheet   gelatin    on    dry 
basis. 

7.  Warm  on  water  bath,  stirring  till  peptone  and 
gelatin  are  dissolved  and  not  allowing  the 
temperature  to  rise  above  60°  C. 

8.  To  500  gm.  of  the  meat  infusion 

add  500  c.c.  of  the  3   per  cent 
agar,    keeping    the    temperature 
below  60°  C. 

9.  Titrate,   after  boiling  one  minute  to  expel 
carbonic  acid. 

10.  Adjust  reaction  to  +1.0  per  cent  by  adding 
normal  hydrochloric  acid  or  sodium  hydrate 
as  required. 

11.  Heat  over  boiling  water  (or  steam)  bath  for 
40  minutes. 

12.  Restore  loss  by  evaporation. 

13.  Readjust  to  +1.0  per  cent  if  necessary  and 
boil  5  minutes  over  free  flame,  constantly 
stirring. 

14.  Make  up  loss  by  evaporation. 


272  APPENDIX 

15.  Filter  through  absorbent  cotton  and  cotton 
flannel,  passing  the  nitrate  through  the  filter 
until  clear. 

16.  Titrate  and  record  the  final  reaction. 

17.  Tube,  using  10  c.c.  of  medium  in  each  tube. 

1 8.  Sterilize  15  minutes  in  the  autoclave  at  120 
degrees,  or  for  30  minutes  in  streaming  steam 
on  three  successive  days.     Put  the  gelatin  at 
once  into  ice-water  till  solidified. 

19.  Store  in  the  ice-chest  in  a  moist  atmosphere, 
to  prevent  evaporation. 


LACTOSE   LITMUS  AGAR 

Lactose  litmus  agar  shall  be  prepared  in  the  same  manner 
as  nutrient  agar,  with  the  addition  of  i  per  cent  of  lactose 
to  the  medium  just  before  sterilization.  The  reaction  shall 
be  made  neutral  to  phenolphthalein  (see  p.  267). 

If  the  medium  is  to  be  used  in  tubes  the  sterilized  azo- 
litmin  solution  shall  not  be  added  until  just  before  the 
final  sterilization. 

If  the  medium  is  to  be  used  in  Petri  dishes  the  steril- 
ized azolitmin  solution  shall  not  be  added  to  the  medium 
until  it  is  ready  to  be  poured  into  the  dishes. 

More  colonies  and  better  general  results  are  obtained 
on  the  lactose  litmus  agar  plates,  when  the  litmus  and 
lactose  are  each  sterilized  separately  and  added  to  the 
plate  with  the  neutral  agar  at  the  time  of  planting.  Good 
results  can,  however,  be  obtained,  if  the  agar  and  lactose 
are  mixed  and  sterilized  in  an  autoclave  at  120°  C.  for 
15  minutes  only. 

The  azolitmin  on  the  market  varies  considerably,  much 
of  that  sold  being  entirely  unreliable  for  the  purpose.  A  i 
per  cent  solution  of  Kahlbaum's  azolitmin,  if  boiled  for 
5  minutes,  readily  dissolves  and  needs  no  correction  for 
acidity  if  added  to  standard  agar.  Many  bacteriologists 
prefer  pure  litmus  to  azolitmin,  and  it  is  therefore  sug- 
gested that  its  use  be  made  optional.  Both  total  and 
red  colonies  may  be  counted  after  from  18  to  24  hours 


APPENDIX  273 

when  incubated  at  37°  C.     Such  tests  are  sometimes  used 
in  the  control  of  nitration  plants. 

LIVER   BROTH 

This  medium  is  made  from  a  hot  infusion  of  beef  liver 
instead  of  fresh  meat,  and  is,  in  other  respects,  with  the 
exception  that  phosphate  is  added  the  same  as  dextrose 
broth,  but  it  is  a  richer  food  medium  for  bacteria.  It 
gives  gas  formation  with  all  species  which  ferment  dextrose 
and  develops  attenuated  bacteria,  whether  gas-forming  or 
not,  to  a  better  degree  than  does  beef  broth.  It  is  also 
especially  suited  to  the  rejuvenation  of  species  in  pure 
culture. 

FORMULA 

Beef  Liver 500  gm. 

Peptone  (Witte's) 10  gm. 

Dextrose 10  gm. 

Di-Potassium  Phosphate  (K^HPO-i) i  gm. 

Water 1000  gm. 

1.  Chop  500  gm.  of  beef  liver  into  small  pieces  and  add 
1000  c.c.  of  distilled  water.     Weigh  the  infusion  and  container. 

2.  Boil  slowly  for  2  hours  in  a  double  boiler,  starting 
cold  and  stirring  occasionally. 

3.  Make  up  any  loss  in  weight  by  evaporation  and  pass 
through  a  wire  strainer. 

4.  To   the  filtrate   add   10  gm.   of  peptone,    10  gm.   of 
dextrose  and  i  gm.  of  potassium  phosphate. 

5.  After  warming  this  mixture  in  a  double  boiler  and 
stirring  it  for  a  few  minutes  to  dissolve  the  ingredients, 
titrate  with  N/2O  sodium  hydrate,  using  phenolphthalein  as 
an  indicator,  and  neutralize  with  normal  sodium  hydrate. 

6.  Boil   vigorously   for   30   minutes   in   a   double  boiler, 
and  5  minutes  over  a  free  flame  with  constant  stirring  to 
prevent  the  caramelization  of  the  dextrose. 

7.  Make  up  the  loss  in  weight  by  evaporation  and  filter 
through  cotton  flannel  and  filter  paper. 


274  APPENDIX 

8.  Tube  and  sterilize  in  an  autoclave  for  15  minutes 
at  120°  C.  (15  pounds). 

Other  valuable  liver  media  (for  use  in  the  identification 
of  B.  sporogenes  and  other  species)  are  prepared  as  given 
below: 

LIVER   GELATIN 

1.  Proceed  as  in  steps  i,  2,  3,  in  preparing  liver  broth. 

2.  Cool  the  filtrate  to  50°  C.     Add  10  per  cent  of  sheet 
gelatin  and  stir  a  few  minutes  until  dissolved. 

3.  Add   i   per  cent  of  peptone,   i   per  cent  of  dextrose 
and  o.i  per  cent  of  potassium  phosphate. 

4.  Stir  until  the  ingredients  are  dissolved,  keeping  the 
temperature  below  50°  C.,  and  then  proceed  as  in  steps 

5,  6,  7,  8. 

LIVER   AGAR 

1.  Chop  500  gm.  of  beef  liver  into  small  pieces,  add  500 
c.c.  of  distilled  water,  and  boil  slowly  for  2  hours,  stirring 
occasionally. 

2.  Add  5  gm.  of  agar  (dried  at  105°  C.  for  30  minutes) 
to  500  c.c.  of  distilled  water  and  digest  for  30  minutes  in 
an  autoclave  at  120°  C.  (15  pounds  pressure). 

3.  After  making  up  the  loss  by  evaporation,  pass  the 
liver  infusion  through  a  wire  strainer,  add  500  c.c.  of  the 
filtrate  to  the  agar  solution  and  proceed  as  in  steps  4,  5, 

6,  7,  8,  in  preparing  liver  broth. 

It  is  very  important  to  note  that  liver  broth  should  not 
be  exposed  to  the  high  temperature  attained  in  the  auto- 
clave any  longer  than  15  minutes,  as  prolonged  heating 
above  the  boiling-point  causes  caramelization  of  the  carbo- 
hydrates, rendering  the  medium  less  delicate  for  bacterial 
development.  For  the  rejuvenation  of  attenuated  cultures, 
especially  B.  sporogenes,  the  addition  of  very  small  pieces 
of  liver  tissue,  which  have  been  sterilized  in  Petri  dishes 


APPENDIX  275 

in  the  autoclave  for  15  minutes  improves  the  rejuvenating 
properties  of  the  medium.  They  should  be  added  to  the 
tubes  after  sterilization. 

Bacterial  growth  being  very  rapid  in  this  medium,  pre- 
liminary rejuvenation  at  37°  C.  should  be  concluded  between 
6  and  12  hours. 

HESSE   AGAR 

Agar  (absolutely  dry) 4.5  gm. 

Peptone,  Witte 10     gm. 

Liebig's  Extract  of  Beef 5     gm. 

Salt 8.5  gm. 

Distilled  Water 1000    c.c. 

Dissolve  4.5  gm.  of  dry  agar  in  500  c.c.  distilled  water 
by  heating  over  a  free  flame,  making  up  loss  in  weight  by 
evaporation.  Into  another  vessel  500  c.c.  of  distilled  water 
is  poured  and  to  this  is  added  10  gm.  of  peptone,  5  gm.  of 
Liebig's  Beef  Extract,  and  8.5  gm.  of  salt.  This  is  heated 
until  all  is  dissolved  and  the  loss  in  weight  by  evaporation 
is  made  up  by  adding  distilled  water. 

Add  the  two  solutions  together;  boil  30  minutes;  make 
up  loss  in  weight  with  distilled  water,  filter  through  absorb- 
ent cotton  held  in  the  funnel  by  cotton  flannel,  passing 
the  filtrate  through  several  times  until  perfectly  clear. 
Test  the  reaction;  adjust,  if  necessary,  to  +1.0  per  cent 
normal  acid,  and  tube,  using  10  c.c.  of  medium  in  each 
tube.  Sterilize  for  15  minutes  at  120°  C.  (15  pounds 
pressure)  in  an  autoclave.  Cool  with  running  water  and 
store  in  an  ice-chest  the  air  of  which  is  saturated  with 
moisture. 

CONRADI-DRIGALSKI   MEDIUM 

These  authors  have  modified  lactose  litmus  agar  by 
adding  to  it  nutrose  and  crystal  violet  and  by  using  3  per 
cent  of  agar  instead  of  i  per  cent.  The  crystal  violet 
strongly  inhibits  the  growth  of  many  other  bacteria,  especially 
cocci,  which  would  also  color  the  medium  red;  the  3  per 


276  APPENDIX 

cent  agar  makes  the  diffusion  of  the  acid  which  is  formed 
more  difficult. 

Three  pounds  of  chopped  beef  are  allowed  to  stand  24 
hours  with  2  liters  of  water.  The  meat  infusion  is  boiled 
one  hour  and  filtered.  Twenty  gm.  of  Witte's  peptone, 
20  gm.  of  nutrose,  and  10  gm.  of  NaCl  are  then  added, 
and  the  mixture  boiled  another  hour.  After  filtration  and 
the  addition  of  60  gm.  of  agar  the  mixture  is  boiled  for  3 
hours,  made  alkaline  and  filtered.  In  the  meantime  300 
c.c.  of  litmus  solution  (Kahlbaum)  are  boiled  for  15  minutes 
with  30  gm.  of  lactose.  Both  solutions  are  then  mixed 
and  the  mixture,  which  is  now  red,  made  faintly  alkaline 
with  10  per  cent  soda  solution.  To  this  feebly  alkaline 
mixture  4  c.c.  of  hot  sterile  10  per  cent  soda  solution  are 
added  and  20  c.c.  of  a  sterile  solution  (i  to  1000)  of  crystal 
violet  (Hochst  B.). 

ENDO  MEDIUM 

Make  3  per  cent  agar  as  follows: 

1.  To  i  liter  of  cold  water  add  30  gm.  of  powdered  agar 
by  sifting  successive  small  portions  upon  the  surface  and 
allowing  them  to  settle. 

2.  Add  10  gm.  of  Witte's  peptone  and  5  gm.  of  Liebig's 
meat  extract. 

3.  Heat  in  double  boiler  until  the  ingredients  are  thoroughly 
dissolved. 

4.  Neutralize  the  sodium  carbonate,  using  litmus  as  an 
indicator,  and  then  add  10  c.c.  of  a  10  per  cent  solution 
of  sodium  carbonate. 

5.  Store  the  medium  in  flasks  in  lots  of  100  c.c.  or  in  larger 
known   quantities,   the  flasks  being   large   enough  for   the 
other  ingredients  to  be  added  later. 

6.  Sterilize  2  hours  in  streaming  steam. 

It  is  essential  to  use  powdered  agar  and  cold  water, 
since  the  agar  settles  more  readily  in  cold  water. 


APPENDIX  277 

To  use  Endo  medium:  (a)  make  a  10  per  cent  solution 
of  sodium  sulphite  in  water;  (b)  make  a  10  per  cent  solution 
of  basic  fuchsin  in  96  per  cent  alcohol;  (c)  add  2  c.c.  of 
the  fuchsin  solution  to  10  c.c.  of  the  sulphite  solution,  pre- 
pared as  above,  and  steam  a  few  minutes  in  an  Arnold 
sterilizer. 

7.  Add  i  gm.  of  chemically  pure  lactose  to  each  100  c.c. 
of  Endo  medium. 

8.  Melt  the  Endo  medium  in  streaming  steam  and  add 
|  c.c.  of  the  fuchsin-sulphite  solution  described  above. 

9.  Pour  plates,   and  allow   them   to   harden   thoroughly 
in  the  incubator. 

The  lactose  used  must  be  chemically  pure,  and  the  sul- 
phite solution  must  be  made  up  fresh  every  day. 

HISS'  MEDIA 

Two  media  are  used:  one  for  the  isolation  of  the  typhoid 
bacillus  by  plate  culture,  and  one  for  the  differentiation 
of  the  typhoid  bacillus  from  all  other  forms  in  pure  culture 
in  tubes. 

Plate  Medium.  The  plate  medium  is  composed  of  10 
gm.  of  agar,  25  gm.  of  gelatin,  5  gm.  of  sodium  chloride, 
5  gm.  of  Liebig's  extract,  10  gm.  of  dextrose,  and  1000  c.c. 
of  water.  When  the  agar  is  thoroughly  melted  the  gelatin 
is  added  and  completely  dissolved  by  a  few  minutes'  boiling. 
The  medium  is  then  titrated,  to  determine  its  reaction, 
phenolphthalein  being  used  as  an  indicator.  The  requisite 
amount  of  normal  hydrochloric  acid  or  of  sodium  hydrate 
solution  is  added  to  bring  it  to  the  desired  reaction;  i.e., 
H-2.  To  clear  the  medium  add  the  whites  of  one  or  two 
eggs,  well  beaten  in  25  c.c.  of  water,  boil  for  45  minutes, 
and  filter  through  a  thin  filter  of  absorbent  cotton.  Add 
the  dextrose  after  clearing.  The  reaction  of  the  medium 
is  most  important;  it  should  contain  never  less  than  2  per 
cent  of  normal  acid. 


278  APPENDIX 

Tube  Medium.  The  tube  medium  contains  agar  5  gm., 
gelatin  80  gm.,  sodium  chloride  5  gm.,  meat  extract  5  gm., 
and  dextrose  10  gm.  to  the  liter  of  water;  reaction  +1.5. 
The  mode  of  preparation  is  the  same  as  for  the  plate  medium, 
care  being  taken  always  to  add  the  gelatin  alter  the  agar 
is  thoroughly  melted,  so  as  not  to  alter  this  ingredient  by 
prolonged  exposure  to  high  temperature.  The  dextrose  is 
added  after  clearing. 

MILK 

The  milk  to  be  used  as  a  culture  medium  shall  be  as 
fresh  as  possible,  "  Certified  Milk  "  being  ordinarily  the 
best  obtainable  in  city  laboratories.  It  shall  be  placed  in 
a  refrigerator  over  night  to  allow  the  cream  to  rise  and 
the  suspended  matter  to  settle.  The  skimmed  milk  shall 
be  siphoned  off  into  a  flask  for  use.  It  will  be  found  more 
convenient,  however,  to  allow  the  milk  to  stand  in  a  separa- 
tory  funnel.  Should  the  milk  be  too  acid  the  reaction  shall 
be  corrected  to  +i  by  the  addition  of  normal  sodium  hydrate. 
It  is  then  ready  to  be  tubed  and  sterilized.  Litmus  milk 
shall  be  prepared  as  above,  with  the  addition  of  sterile  i 
per  cent  azolitmin.  As  it  is  impossible  to  make  each  lot 
of  litmus  milk  with  the  same  shade  of  color,  it  is  recom- 
mended that  a  control  tube  be  always  exposed  with  the 
inoculated  tubes  for  purposes  of  comparison. 

NITRATE   BROTH 

Dissolve  i  gm.  peptone  in  i  liter  of  tap  water,  and  add 
0.2  gm.  of  nitrite-free  potassium  nitrate. 

PEPTONE  SOLUTION  FOR  INDOL  TEST 

Broth  which  has  been  inoculated  with  B.  coli  to  remove 
the  muscle  sugar  contains  toxins  which  interfere  with  the 
proper  growth  of  many  species,  hence  peptone  solution, 


APPENDIX  279 

(i  per  cent  peptone  in  water)  is  recommended  for  use  in 
the  indol  test. 

^ESCULIN   MEDIA 

Broth.  Dissolve  10  gm.  peptone,  5  gm.  commercial 
sodium  taurocholate  (or  glycocholate),  o.i  gm.  aesculin  and 
0.5  gm.  soluble  iron  citrate  in  i  liter  distilled  water. 

Tube  in  5  c.c.  quantities  in  test-tubes  and  sterilize. 

It  is  preferable  to  keep  the  iron  citrate  solution  separate 
and  to  add  it  to  the  rest  of  the  media  as  used,  since  the 
iron  causes  a  precipitate.  Sometimes  the  iron  citrate  will 
not  readily  dissolve.  In  such  case  a  few  drops  of  ammonia 
will  cause  immediate  solution,  especially  on  warming.  The 
excess  of  ammonia  should  then  be  boiled  off. 

A  gar.  This  medium  is  made  in  the  same  manner  as  the 
broth  with  the  addition  of  the  usual  amount  of  agar. 

APPARATUS 

Few  definite  requirements  need  be  made  respecting 
apparatus.  The  quality  of  the  glass  shall  be  such  as  not 
to  be  easily  acted  upon  by  the  reagents  used,  and  all  glass- 
ware shall  be  scrupulously  clean  when  used.  When  nec- 
essary it  shall  be  sterilized  by  dry  heat  for  one  hour  at 
about  150°  C.  A  slight  browning  of  the  cotton  stoppers 
is  a  good  index  of  proper  exposure. 

In  some  operations,  as  for  example,  the  determination 
of  the  thermal  death  point,  it  is  necessary  to  use  test-tubes 
of  a  definite  size  and  thickness.  For  this  purpose  the 
standard  size  culture  tube  shall  be  15  cm.  long,  1.6  cm. 
in  diameter,  and  of  medium  weight.  Tubes  to  be  filled 
with  gelatin  for  quantitative  work  may  be  those  described 
as  6  in.  Xf  in.  "  heavy." 

The  standard  loop  for  making  transfers  shall  be  pre- 
pared as  follows: 


280  APPENDIX 

Bend  the  end  of  a  piece  of  No.  27  platinum  wire  about 
10  cm.  long  over  a  bit  of  No.  10  wire,  and  fasten  the  loop 
thus  formed  into  a  glass  rod  to  serve  as  a  handle.  A 
loopful  of  culture  shall  be  interpreted  as  meaning  all  the 
fluid  that  the  loop  can  hold.  That  is,  the  fluid  shall  form 
a  bi-convex  body  and  shall  not  be  simply  a  film  covering 
the  space  in  the  loop. 

The  standard  fermentation  tube  shall  be  a  tube  1.5  cm. 
in  diameter,  bent  at  an  acute  angle,  closed  at  one  end  and 
provided  with  a  bulb  at  the  other  which  is  large  enough 
to  receive  all  the  liquid  contained  in  the  closed  portion. 
The  length  of  the  closed  end  of  the  tube  shall  be  about 
14  cm.  The  bulb  shall  have  a  diameter  of  about  3.8  cm. 

INCUBATION 

There  shall  be  two  standard  temperatures  of  incubation— 
20°  C.  and  37°  C.,  the  first  corresponding  to  ordinary  room 
temperature,  the  second  to  blood  heat.  The  temperature 
of  the  incubators  shall  not  be  allowed  to  vary  from  these 
two  standards  by  more  than  i°  C.  in  either  direction. 

The  atmosphere  of  the  incubator  shall  be  kept  moist, 
preferably  near  the  point  of  saturation.  The  incubator  shall 
be  ventilated  so  as  to  insure  a  reasonably  good  circulation 
of  air  in  order  to  prevent  the  accumulation  in  the  incubator 
of  gases  which  might  be  prejudicial  to  the  development  of 
the  bacteria. 

No  definite  period  of  incubation  can  be  prescribed  which 
will  be  suitable  for  all  the  work  of  species  determination, 
but  in  reporting  results  the  period  used  shall  always  be 
stated  and  form  a  part  of  the  report.  General  statements 
as  to  the  necessary  periods  will  be  found  in  connection 
with  the  principal  tests. 


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WILLSON,  H.  S.  1905.  The  Isolation  of  B.  Typhosus  from  Infected 
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WINSLOW,  C.-E.  A.  1901.  The  bacteriological  Examination  of 
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INDEX   OF  AUTHORS 


Abba,  144,  161. 

Calmette,  235,  241. 

Abbott,  36. 

Cambier,  36,  47. 

Adami,  81. 

Cameron,  245. 

Adams,  30,  44,  45,  46. 

Carpenter,  237. 

Altschuler,  83. 

Chick,  65,  105,  153. 

American  Committee  on  Standard 

Chopin,  81. 

Methods,  136,  137,  193,  208. 

Christian,  120. 

Amyot,  142. 

Clark,  H.  W.,    57,    72,    m,  146, 

157,  162,  166,  242,  248,  251. 

Baker,  203,  204. 

Clark,  W.  M.,  118,  199. 

Barthel,  147. 

Clemesha,  17,    21,   108,   114,   H5> 

Baton,  107,  in,  117. 

184,  193,  210. 

Bachmann,  64,  231. 

Cramer,  54. 

Beckmann,  143. 

Conn,  245. 

Belcher,  213. 

Conradi,  76. 

Belitzer,  140. 

Copeland,  102,  105,  192. 

Bergey,  180,  191. 

Bettencourt,   141,   142,   147,   172. 

Biffi,  176. 

Davis,  118,  199. 

Blachstein,  144. 

Deehan,  180,  191. 

Blunt,  16. 

Dibdin,  228. 

Bolton,  53. 

Ditthorn,  85. 

Borges,  141,  142,  147. 

Doebert,  77. 

Boullanger,  241. 

Dolt,  130. 

Braun,  122. 

Downes,  16. 

Brezina,  55. 

Drew,  13,  36. 

Brotzu,  140. 

Drigalski,  76,  85. 

Brown,  151. 

Duclaux,  6. 

Bmns,  144. 

Duggeli,  147- 

Buchan,  248. 

Dunbar,  97. 

Buchner,  17. 

Dunham,  56,  96. 

Bulstrode,  225,  248. 

Durham,  93,  95. 

Burri,  150,  183. 

Dyar,  140. 

307 


308 


INDEX  OF  AUTHORS 


Eddy,  232. 

Egger,  26. 

Eijkman,  120. 

Ellms,  36. 

Eisner,  75. 

Endo,  76. 

English  Committee,  136. 

Escherich,  99. 

Eyre,  140. 

Fehrs,  15. 

Ferguson,  116. 

Ferreira,  102,  141. 

Ficker,  80,  84. 

Fischer,  2,  26,  90. 

Flatau,  90. 

Fox,  91. 

Frankland,  12,  37,  98. 

Fremlin,  140. 

Freudenreich,  16,  150. 

Fromme,  113,  119,  121,  142,  163, 

171,  225. 
Frost,  1 6,  179. 
Fuller,  16,  32,  43,  57,  116,  182. 

Gaehtgens,  77. 

Gage,  8,  9,  30,  31,  43,  44,  45,  46, 
69,  70,  72,  107,  108,  109,  in, 
122,  133,  146,  157,  162,  166, 
177,  186,  187,  203,  218,  230, 
242,  249. 

Garre,  15. 

Gartner,  14,  35,  53,  159,  170,  171. 

Gautie,  172. 

Gildemeister,  85. 

Gordan,  147,  209. 

Gorham,  249,  254,  256. 

Hachtel,  79,  91,  97. 
Hale,  116,  126,  127,  129. 
Ham,  244,  249,  250,  264. 
Hammerl,  151. 
Hammond,  113,  115,  116. 


Hankin,  75. 

Hansen,  221. 

Harrison,  129. 

Hazen,  19. 

Heider,  226. 

Heraeus,  35. 

Hesse,  29,  45,  47. 

Hilgermann,  120. 

Hill,  36,  64. 

Hiss,  78,  89. 

Hoffmann,  24,  80. 

Holman,  213. 

Hoover,  102,  192. 

Horder,  199,  209. 

Horhammer,  15.    . 

Horrocks,  15,  26,  87,  153,  182,  203. 
207,  212. 

Horta,  102,  141. 

Houston,  n,  21,  22,  24,  27,  96, 
115,  141,  146,  150,  152,  158, 
159,  162,  178,  185,  189,  197, 

202,     207,      209,      213,     222,     236, 
239- 

Howe,  175,  177,  180-199. 
Hunnewell,  115,  118,  203. 
Huntemiiller,  15. 

Irons,  106,  109,  120,  122. 

Jackson,  64,  78,  81,  91,  122,  123, 

131,  192. 
Janowski,  6. 

Johnson,  58,  142,  182,  235,  232. 
Jordan,  14,  16,  17,  20.  23,  39,  55, 

153,  155,  226. 

Kabrhel,  25. 

Kaiser,  163. 

Keith,  140. 

Kellerman,  26,  238,  240. 

Kimberly,  238,  240. 

Kinnicutt,  234,  236. 

Kisskalt,  9. 


INDEX  OF  AUTHORS 


309 


Kloumann,  80. 

Klein,  75,  81,  146,  211,  213. 

Kligler,  148. 

Klotz,  82,  213. 

Koch,  97. 

Kohn,  38,  53. 

Konradi,  24. 

Konrich,  101,  102,  121,  141,  147, 

148,    163,    169,    170,    171,    176, 

183,  187,  188. 
Korschun,  15. 
Kranepuhl,  239 
Kruse,  143,  169. 
Kiibler,  90. 
Kurpjuweit,  239. 

Laws,  92,  202. 

Lederer,  64,  231. 

LeGros,  203. 

Lemke,  77. 

Lentz,  77. 

Levy,  144. 

Lochridge,  19. 

Loeffler,  77,  96. 

Longley,  107,  in,  117,  165. 

Lubenau,  87. 

MacConkey,   122,   175,   177,   178, 

180,  184,  189,  190,  191. 
Makgill,  122. 
Marshall,  176. 
Marvel,  246. 
Maschek,  26,  35. 
Mass.  State  Board  of  Health,  136, 

161. 

Massol,  241. 
Massini,  183. 
Mathews,  66,  105. 
Mayer,  A.,    18,  19. 
Mayer,  G.,  56. 
McWeeney,  175. 

Melia,  81,  91,  116,  126,  127,  129. 
Miquel,  6,  37,47,  Si,  55- 


Moore,  140. 

Moroni,  144. 

Muer,  131. 

Muller,  30,  31,  36,  84,  183. 

Neufeld,  90. 
Neuman,  13,  120,  147. 
Newlands,  244,  249,  250,  264. 
Nibecker,  12,  67,  68,  in,  133,  204. 
Niedner,  45,  47. 
Nieter,  85. 
Nowack,  77,  121. 
Nuttall,  211. 

Orlandi,  161. 
Osgood,  226. 
Otto,  13. 

Pakes,  153. 

Palmer,  209. 

Papasotiriu,  146. 

Paredes,  102,  141. 

Parietti,  74. 

Park,  89. 

Pease,  249. 

Peckham,  182. 

Penfold,  183. 

Petruschky,  168. 

Phelps,  30,  31,  43,  44,  71,  in,  113, 

115,    116,    122,    133,    177,    186, 

187,  233,  237,  249,  252. 
Philbrick,  10,  n,  54. 
Poujol,  144. 

Pratt,  234,  236,  238,  240. 
Prescott,  12,  26,  43,  67,  107,  146, 

203,  204. 
Procaccini,  17. 
Pusch,  168. 

Rapp,  17,  18. 
Refik,  143. 
Reinsch,  43. 
Remlinger,  93. 


310 


INDEX  OF  AUTHORS 


168 


Reynolds,  119. 
Rickards,  248. 
Rideal,  66,  229,  237,  242. 
Riedel,  38. 
Rivas,  1 80. 
Rogers,  118,  199. 
Rondelli,  161. 
Roth,  80. 
Rothberger,  122. 
Ruediger,  22,  23. 
Russell,  13,  16. 

Sauerbeck,  183. 

Savage,  24,  62,  122,  141,  145 

214,  225. 
Sawin,  124,  125. 
Schepilewski,  83. 
Scheurlen,  14. 
Schneider,  93. 
Schottelius,  96. 
Schuder,  84. 

Schultz-Schultzenstein,  241. 
Schumacher,  239. 
Sedgwick,  4,  21,  43,  95,  105. 
Shell  Fish  Commission,  A.P.H.A 

255- 

Shuttleworth,  59. 
Smith,  101,  106,  140,  150,  180. 
Soper,  246. 
Spitta,  14. 
Stamm,  117. 
Starkey,  86,  95,  96. 
Sternberg,  52. 
Stiles,  248. 

Stokes,  79,  91,  97,  122,  127. 
Stokvis,  15. 
Stoner,  127. 
Swellengrebel,  15. 


Thoman,  75. 
Thorne-Thorne,  246. 
Thomson,  75. 
Thresh,  36,  95,  246. 
Tiemann,  14,  35. 
Tietz,  77. 
Tully,  73. 
Twort,  183. 

Vallet,  84. 

Van  Buskirk,  50. 

van  der  Leek,  129. 

Vincent,  56,  172,  213,  223. 

Walker,  101,  147,  180,  232. 

Wathelet,  92. 

Weissenfeld,  145. 

Welch,  211. 

West,  180. 

Wheeler,  18. 

Wherry,  178. 

Whipple,  18,  19,  38,  39,  41,  44,  46, 
63,  71,  72,  no,  118,  181,  221. 

Whittaker,  26. 

Widal,  89. 

Wilhelmi,  248. 

Willson,  80,  84,  90. 

Winslow,  12,  19,  21,  29,  30,  34, 
60,  66,  67,  68,  71,  101,  in,  118, 
133,  147,  148,  155,  !8o,  199-  203, 
209,  213,  221,  232,  233,  234,  236. 

Wolffhugel,  26,  38. 

Woodman,  221. 

Wright,  140. 

Wurtz,  65,  104. 

Zagari,  15. 
Zeit,  16,  23. 


SUBJECT   INDEX 


Acid-forming  organisms,  65. 
Acid  wastes,  antiseptic  effect   of, 

20. 

Aesculin,  129. 
Aesculin  bile  medium,  129. 
Aesculin  medium,  preparation  of, 

279. 

Aesculin  test,  129. 
Agar,  drying  of,  78. 
Agar,  for  body  temperature  count, 

63. 

Agar,  preparation  of,  270. 
Agglutination  of  typhoid  bacilli, 

81,  82,  83. 

Anaerobic  bacteria,  201. 
Anaerobic    spore-forming    bacilli, 

211. 

Anglo-American  procedure,  172. 
Aniline  dyes,  use  of,  77. 
Antagonism,  16. 

Anthrax,  occurrence  in  water,  98. 
Apparatus,  treatment  of,  279. 
Arbitrary  standards,  fallacy  of,  51. 
Atmospheric  waters,  5. 
Atypical  colon  bacilli,  135. 
Azolitmin,  272. 

Bacillus,  anaerobic  spore-forming, 

211. 

Bacillus  acidi  lactici,  189. 
Bacillus  aerogenes,  189. 
Bacillus  alcaligenes,  94. 


Bacillus  anthracis,  98. 
Bacillus  coli,  94,  99. 

cultural  features,  100. 

discovery,  99. 

distribution  in  waters,  152. 

effect  of  temperature  on,  113. 

fermentation  reactions,  101. 

importance  of  numbers,  149. 

index  of  pollution,  219. 

in  cold-blooded  animals,  142. 

in  ground  waters,  113. 

in  sewage,  230. 

in  soils,  152. 

isolation  of,  104,  132. 

isolation   by   Conradi-Drigalski 
medium,  106. 

isolation  by  Endo  medium,  106. 

isolation    by    synthetic    media, 
130. 

mutations  in,  183. 

occurrence,  99. 

occurrence  in  animals,  140,  141. 

occurrence  on  plants,  146. 

pathogenicity,  100. 

positive  isolations,  no. 

preliminary  enrichment  of,  106. 

quantitative  test,  138. 

standard  tests  for,  115. 

streak  characteristics,  134. 

ubiquity  of,  143. 

Bacillus  coli  and  sewage  strepto- 
cocci, 205,  206. 

311 


312 


SUBJECT  INDEX 


B.  communis,  181. 
B.  communior,  181. 
B.  dysenteriae,  89,  94. 
B.  enteritidis,  94. 
B.  mycoides,  133. 
B.  sporogenes,  211. 

characteristics  of,  213. 

growth  in  liver  broth,  212. 

in  sewage,  213. 

isolation  of,  211. 

occurrence  of,  212. 
B.  typhi,  94. 

identification  of,  87. 

comparison  with  B.  coli,  87. 

in  oysters,  91. 

isolation  from  water,  89,  90. 

isolation  of,  90. 

small  numbers  in  water,  92. 
B.  welchii,  129,  134,  138,  211. 
Bacteria,  affected  by  temperature, 

20. 

as  agents  of  decomposition,  3. 

count  of  as  index  of  efficiency 
of  niters,  59. 

counts  of  on  different  media,  44. 

development  at  high  tempera- 
tures, 69. 

distribution,  i. 

diurnal  variation  of  in  sewage, 

233- 
effect  of  composition  of  media 

on,  19. 

effect  of  light,  16,  17. 
effect  of  rainfall  on,  7. 
effect  of  storage  on,  10. 
estimation  of,  48. 
expression  of  counts  of,  49. 
factors  influencing  diminution, 

13- 

field  determinations  of,  49. 
food  requirements,  2. 
in  contact  effluents,  240. 
in  disinfected  effluents,  240. 


Bacteria  in  driven  wells,  27. 
in  dust  and  air,  6. 
in  earth,  6. 
in  filtered  waters,  57. 
in  ground  waters,  25. 
in  lakes  and  ponds,  12. 
in  ocean,  13. 
in  oysters,  seasonal  variation  of, 

254,  256. 

in  polluted  streams,  7. 
in  rain  and  snow,  6. 
in  sand  effluents,  234,  240. 
in  septic  effluents,  240. 
in  sewage,  232. 
in  sewage  effluents,  233. 
in  shallow  wells,  26. 
in  springs,  26. 
in  surface  waters,  54. 
in  trickling  filter  effluents,  236. 
in  unpolluted  streams,  7. 
in  water,  5. 

in  water  and  shellfish,  249,  250. 
metatrophic,  2. 

microscopic  enumeration  of,  29. 
mineral  nutrients  for,  53. 
multiplication  in  stored  waters, 

37- 

nitrifying,  4. 

number  of  as  index  of  purity,  60. 

numbers  of  in  sewage,  232. 

occurrence,  i. 

paratrophic,  2. 

pathogenic  in  water,  74. 

proto trophic,  2,  29. 

relation  to  character  of  food,  29. 

quantitative  methods  of  deter- 
mination, 29. 

seasonal  variation  of,  7. 

sedimentation  of,  14. 
Bacteriological     examination     of 

shellfish,  244,  248. 
Bacteriological     examination     of 
sewage,  methods  of,  229. 


SUBJECT  INDEX 


313 


Bacteriological    examinations     of 

water,  significance  of,  217. 
Bacteriological  methods  for  super- 
vision of  filtration  plants,  227. 
Bacteriological  methods  for  super- 
vision of  water  supplies,  227. 
Bacteriological  methods  in  detect- 
ing sewage  distribution,  226. 
Bacteriology  of  sewage,  228. 
Bacteriology  of  sewage  effluents, 

228. 

Bacteriology  of  sewage  filters,  241. 
Bacteriological  examination,  220. 
advantages  of,  220. 
certainty  of,  223. 
delicacy  of,  221. 
Bile,  importance  of,  127. 
Bile  media,  81,  122. 
Bile  salts,  122. 

selective  action  of,  123,  128. 

Body  temperature  count,  42,  61. 

relation  to  hot  weather,  70. 

Caffein,  selective  action  of,  80. 
Calcium  hypochlorite,  238. 
Carbon    dioxide,     absorption   of, 

117. 

Chemical  disinfection,  238. 
Chlorine  disinfection,  relation  to 

counts,  72. 

Cholera  red  reaction,  97. 
Cholera  spirillum,  isolation  from 
water,  96. 

media  for,  96. 
Clams,  244,  263. 
Cold,  action  of  on  bacteria,  21. 
Colon  bacilli,  64,  99. 

as  index  of  pollution,  168. 

as    index    of     self-purification, 

153- 

atypical,  135,  177. 
colonies  of,  133. 
comparison  with  B.  typhi,  87. 


Colon     bacilli,      distribution    in 
waters,  152. 

effect  of  temperature  on,  22. 

"  flaginac,"  185. 

importance  of  numbers,  149. 

increased      survival      in      cold 
weather,  22. 

in  cultivated  soil,  170. 

in  dust,  148. 

in  filter  effluents,  166. 

in  filtered  waters,  163. 

in  foods,  147. 

in  fruits,  147. 

in  grains,  146. 

in  ground  waters,  161. 

in  sewage,  231. 

in  sewage  effluents,  231. 

in  shallow  wells,  162. 

in  shellfish,  252. 

in  soil,  148. 

in  surface  waters,  157. 

in  unpolluted  waters,  155. 

isolation  of,  104. 

isolation  by  bile  media,  122. 

on  plants,  146. 

standard  tests  for,  115. 

"  typical,"  176. 

ubiquity  of,  143. 

varieties  of,  174. 

viability  at  different  tempera- 
tures, 128. 
Colon  group,  99. 

characteristics,  104. 

distribution  in  water,  185. 

Jackson's  classification  of,  192. 

McConkey's     classification    of, 
189. 

statistical  classification,  198. 

tests  for,  174. 

variations  in,  181. 
Colon  test,  101. 
Colon  typhoid  group,  94. 

reactions  of,  95. 


314 


SUBJECT  INDEX 


Comparison  of  sand  and  mechan- 
ical niters,  58. 

Composition    of  medium,  impor- 
tance of,  43. 
Confirmatory  tests,  136. 
Conradi-Drigalski  medium,  76. 

for  B.  coli,  106. 

preparation  of,  275. 
Contact  beds,  235. 
Counting,  48. 
Crystal  violet,  76. 
Culture    media,    ingredients    for, 
265. 

preparation  of,  265. 

reaction  of,  267. 

sterilization  of,  266. 

titration  of,  267. 

uniform  methods  for,  265. 

Deep  wells,  bacteria  in,  27. 
Dextrose  broth,  107. 

advantages  of,  108. 

comparison    with    lactose    bile, 
125. 

disadvantages  of,  108. 
Dextrose  test,  failure  of,  116. 
.  Diluting  samples,  40. 
Disinfection  of  sewage,  237. 
Disinfection   of   sewage   effluents, 

237- 
Distribution    of    types    of    colon 

group  in  waters,  184. 
Division  of  colon  group,  1 74. 
Dunham's  solution,  97. 
Dysentery,  spread  by  water,  95. 

Eijkman  test,  120. 
Endo  medium,  76. 

for  B.  coli,  106. 

preparation  of,  276. 
Examination  of  shellfish,  standard 

methods  for,  255,  257. 
Examination  of  shell  water,  251. 


Expression    of    quantitative    re- 
sults, 49. 

Fermentation  of  lactose,  114. 
Fermentation  test,  105,  109,  179. 

effect  of  temperature  on,  112. 

exceptions  to,  in. 

interpretation  of,  no. 
Field  kits,  49. 
Field  methods,  50. 
Filter  plants,   routine   control   of, 

230. 

Filtered  waters,  bacteria  in,  57. 
Filtration  in  Japan,  58. 
"  Flaginac  "  B.  coli,  185. 
Food  supply,  importance  of,  20. 

Gartner  bacillus,  95. 
Gas-forming    bacteria,  growth  in 

liver  broth,  131. 
Gas  production  in  vacuo,  118. 
Gas  ratio,  100,  109. 

effect  of  age  on,  117. 

unreliability  of,  118. 
Gelatin  liquefaction,  177. 
Gelatin  plates,  use  of,  41. 
Gelatin,  preparation  of,  270. 
Green  plants,  food  requirements, 

3- 

Ground  waters,  6. 
bacterial  content  of,  56. 
B.  coli  in,  113. 

Hesse  medium,  78. 

drying  of,  78. 

preparation  of,  275. 
High    temperatures,     significance 

of,  69. 
Hiss  agar  medium,  78. 

preparation  of,  277. 
Hog  cholera  bacillus,  94. 

Incubation,  46,  280. 


SUBJECT  INDEX 


315 


Incubation  period,  41,  47. 

for  body  temperature  count, 

64. 
Incubator,  necessity  for  moisture 

in,  46. 

Indol  test,  176. 
Infusoria,  destruction  of  bacteria 

by,  15- 

Interpretation  of  results,  51. 
Intestinal  bacilli,  103,  201. 
Isolation  of  B.  coli,  104,  132. 

by  bile  media,  122. 
Isolation  of  cholera  spirillum,  97. 
Isolation  of  streptococci,  203. 

Lactose  bile,  81,  122,  126. 
action  of  bacteria  on,  102. 
advantage  of,  127. 
decomposition  of,  64. 

comparison      with      dextrose 

broth,  125. 

preparation  of,  269. 
Lactose  fermentation,  114. 

importance  of,  115. 
Lactose  fermenting  bacilli,  102. 

effect  of  storage  on,  115. 
"  Lamirascsal,"  streptococci,  210. 
"  Larasacsal  "  streptococci,  210. 
Light,  destructive  effect  on  bac- 
teria, 16,  17. 
Litmus  lactose  agar,  64,  104. 

counts,  65. 

incubation  of,  132. 

preparation  of,  272. 
Liver  agar,  preparation  of,  274. 

broth,  131. 

preparation  of,  273. 
Liver  gelatin,  preparation  of,  274. 

Malachite  green  agar,  77. 
McConkey's     group     in     filtered 
water,  198. 


McConkey's  groups  in  raw  waters, 

198. 
McConkey's    groups     in    stored 

waters,  198. 

Mechanical  filtration,  57. 
Methods  of  estimation,  48. 
Middletown  outbreak,  245. 
Milk,  preparation  of,  278. 
Mussels,  244. 
Mutations  in  B.  coli,  183. 


Nahrstoff  agar,  ratio,  31. 
Nahrstoff  gelatin,  ratio,  31. 
Nahrstoff-Heyden  agar,  29. 
Neutral  red,  122. 
Neutral  red  reaction,  121. 
Neutral  red  test,  178. 
Nitrate  broth,  preparation  of,  287. 
Nitrates,  3. 
Nitrite  test,  177. 
Nitrogen  cycle,  4. 
Nitroso-indol  reaction,  97. 
Nutrient    broth,    preparation    of, 

268. 
Nutrose,  76. 


Obligate  parasites,  29. 
Overgrowth,  effect  of,  119. 
Oysters,  244. 

B.  coli  in,  259. 

bacterial  counts  of,  258. 

bacteriological   examination  of, 

257- 

opened,  examination  of,  262. 
rating  for  B.  coli,  260. 
sampling,  257. 
Oysters  and  typhoid,  246. 


Para-colon  bacilli,  187. 
Para- typhoid  bacilli,  94. 


316 


SUBJECT  INDEX 


Pathogenes  in  water,  74. 

Pathogenic  bacteria,  74. 

Peptone,  importance  of,  44. 

Petri  dishes,  105. 

Phenol  agar,  105. 

Phenol  broth,  119. 

Phenol  dextrose  broth,  108. 

Plate  method,  105. 

Plating,  40. 

Polluted  shellfish,  effect  of  cook- 
ery on,  248. 

Polluted   waters,   isolation   of   B. 
coli  from,  107. 

Pollution,  progressive,  223. 
of  shellfish,  245. 
temporary,  225. 

Porous  tops,  64,  105. 

Precipitation   of    typhoid   bacilli, 
81,  83,  84. 

Preliminary  enrichment,  106. 

Presumptive  test,  no. 

Presumptive  tests,  various,  126. 

Presumptive  tests  with  bile  media, 
124. 

Progressive    pollution,    detection 
of,  223. 

Prototrophic  bacteria,  29. 

Protozoa,    reduction    of    bacteria 
by,  15. 

Pumping,  effect  of  on  bacteria,  35. 


Quantitative    bacteriological    de- 
termination, 29. 
interpretation  of,  51. 

Quantitative    results,    expression 
of,  49. 


Rainfall,  effect  of  on  bacteria,  7. 
Reaction,  importance  of,  43. 
Reaction  of  culture  media,  267. 
Reaction  optimum,  43. 


Reducing  bacteria,  importance  of, 
242. 

Relation  between  room  tempera- 
ture and  body  temperature 
counts,  61,  67,  71. 

Room  temperature  counts,  61. 


Saccharose,  fermentation  of,  180. 
Samples,  dilution  of,  40. 
Samples,  icing  of,  39. 
Sampling,  33. 
Sand  nitration,  57. 
Sanitary    chemical    analysis,    sig- 
nificance of,  216. 
Sanitary  inspection,  215. 
importance  of,  172. 
Seasonal  variation,  72. 
Sedimentation,  7. 
Sedimentation  of  bacteria,  14. 
Self-purification,  20. 
Selective  media,  219. 
Selective  temperatures,  219. 
Sewage,  bacteria  in,  214. 

bacteriology  of,  228. 

colon  bacilli  in,  231. 
Sewage  effluents,  bacteriology  of, 
228. 

colon  bacilli  in,  231. 

standards  for,  239. 
Sewage  examination,   use  of  bile 

medium  in,  231. 
Sewage  sampling,  error  of,  231. 
streptococci,  134,  201. 

as  index  of  pollution,  202. 

isolation  of,  204. 

Sewage  streptococci  and  B.  coli, 
growth  in  dextrose  broth,  205, 
206. 

detection  of,  206. 
Sewage  and  sewage  effluents,  228, 

229. 
Shallow  wells,  bacteria  in,  26. 


SUBJECT  INDEX 


317 


Shallow  wells,  body  temperature 

count  in,  67. 
Shellfish  and  disease,  244. 

bacteriological  examination   of, 
248,  251. 

bacteria  in  shucked,  254. 

careless  handling  of,  253. 

colon  bacilli  in,  252. 

self-purification  of,  252. 

standards  of  interpretation,  264. 

streptococci  in,  252. 
Shell  water,  examination  of,  251. 
Significance  of  37°  count,  62. 
Specific   sewage   organisms,    tests 

for,  230. 

Springs,  bacteria  in,  26. 
Standard  msthods,  32,  33,  41. 

necessity  of,  45. 
Standard  reaction,  268. 
Standards    for    sewage    effluents, 

239- 

Staphylococci  in  sewage,  202. 
Storage,  effectiveness  of,  25. 

effect  of  on  bacteria,  10,  21. 

effect  of  duration  of,  23. 

effect  on  lactose  fermenters,  115. 
Storage  of  samples,  effect  of,  37. 
Stored  waters,  6. 
Streptococci,  64,  133. 

antagonism  to  colon  bacilli,  208.  | 

comparative  fermentations  by, 
210. 

from  different  animals,  209. 

in  sewage,  202. 

in  saliva,  204. 

in  shellfish,  252. 

in  polluted  waters,  203. 

in  stored  sewage,  207. 

indicative   of   recent  pollution, 
207. 

index  of  pollution,  220. 

isolation  of,  203. 

on  animal  bodies,  204. 


Streptococci,    varieties    of,     208, 

209. 

Streptococci,  "  lamiracsal,"  210. 
Streptococci,  "  larasacsal,"  210. 
Streptococcus  equinus,  209. 
Sugar  broths,  preparation  of,  269. 
Sugar  reactions,  179. 
Sugars,  action  of  bacteria  on,  102. 
Surface  waters,  5. 

bacterial  content  of,  54. 
Swimming  pools,  bacteria  in,  72. 
Synthetic  media,  130. 


Temperature,  effect  on  B.  coli,  113. 

effect  on  bacteria  in  water,  20. 

effect  on  fermentation  test,  112. 
Temporary  pollution,  detection  of, 

225. 

Titration  of  culture  media,  267. 
Toxic  products,  effect  of,  15. 
Trickling  filters,  235. 
Typhoid,      occurrence      in      cold 

weather,  22. 

Typhoid  and  shellfish,  245. 
Typhoid  bacilli,  agglutination  of, 
81,  82,  83. 

artificial     infection     of     water 
with,  24. 

developing  on  malachite  green 
media,  77. 

effect  of  oxygen  on,  19. 

enrichment  in  caffein  media,  79. 

enrichment  of,  75. 

examination  of  water  for,  74. 

in  polluted  waters,  23. 

in  pure  culture,  16. 

in  tap  water,  24. 

in  unsterilized  waters,  23. 

isolation  by  lactose  bile,  81. 

isolation  of,  7.6. 

media  for,  76,  77,  78,  79. 

precipitation  of,  81,  83,  84. 


318 


SUBJECT  INDEX 


Typhoid,  preliminary  enrichment 
of,  79. 

separation  by  motility,  85. 

small  numbers  in  water,  92. 

summary  of  isolation  methods, 
86. 

uncultivated  strains  in  water,  24. 

viability  in  mud,  24. 

viability  in  sewage,  16. 

viability  in  water,  '15. 
"  Typical"  B.  coli,  176. 


Unpolluted  waters,  body  tempera- 
ture count  in,  68. 
Urea,  decomposition  of,  3. 

Voges-Proskauer  reaction,  180. 

Waters,  classification  of,  5. 
Wells,  bacteria  in,  26,  56. 

B.  coli  in,  26,  162,  163. 

deep,  27. 


